CN113614224A

CN113614224A - Novel method for producing antibody

Info

Publication number: CN113614224A
Application number: CN201980088417.9A
Authority: CN
Inventors: 鲁白; 姚虹洋
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2018-11-08
Filing date: 2019-11-08
Publication date: 2021-11-05
Also published as: WO2020094121A1; US20210388060A1

Abstract

The present disclosure relates to methods of producing antibodies or antigen-binding fragments thereof that specifically bind to an antigen of interest, to methods of inducing PBMC proliferation, B cell activation and differentiation, B cell maturation, and/or promoting class switching in antibody-producing PBMCs to produce IgG, to compositions for in vitro immunization, and to methods of identifying antibody enhancers for in vitro immunization.

Description

Novel method for producing antibody

Technical Field

The present disclosure relates generally to novel methods for producing antibodies, and in particular to methods for producing fully human antibodies in vitro.

Background introduction

Antibody production methods have been widely used in laboratories and clinics. These methods include hybridoma technology, transgenic animal models, and in vitro immunization. Traditional hybridoma technology is a mainstream maturation technology, including immunization of animals, isolation of lymphocytes, fusion of lymphocytes with immortalized cells (e.g., myeloma), antibody humanization, and affinity maturation. Antibodies can be produced in high throughput, but it has to face a number of disadvantages including high cost, long production cycle, low affinity, unpredictability of the variable regions of the heavy and light chains, etc. Transgenic animal models are a relatively new technology that genetically modifies animals to express human antibody variable regions by unknown mechanisms. In recent years, the literature has proposed the concept of in vitro immunization techniques that do not require immunization of animals, which are less expensive to produce, faster and easier to handle, and which antibodies can be fully human without the need for humanization procedures. However, there are few reports of successful antibody production using this approach. Therefore, there is a continuing need to develop new effective in vitro immunization techniques to produce fully human antibodies.

Disclosure of Invention

The present disclosure provides a method of producing an antibody or antigen-binding fragment that specifically binds to an antigen of interest, the method comprising:

mixing the antigen, an antibody-producing cell composition (AGC), and an enhanced antibody-producing composition in a culture medium to form a mixture,

(ii) incubating the mixture in the presence of a nutrient,

obtaining an antibody from the mixture, wherein the antibody is a monoclonal antibody,

wherein the AGC comprises at least one B cell and at least one other type of cell derived from Peripheral Blood Mononuclear Cells (PBMCs), and the antibody enhancing composition comprises one or more adipose tissue-derived secreted proteins (ADSPs).

In certain embodiments, wherein the antibody enhancing composition further comprises IL2 and/or IL 21.

In certain embodiments, wherein the adipose tissue-derived secreted protein comprises one or more cytokines and/or one or more cell adhesion molecules.

In certain embodiments, wherein the cytokine comprises one or more interleukins. In certain embodiments, the interleukin is selected from IL1 β, IL1f9, IL10, IL27, IL33, IL18BP, IL4, IL3, IL5, IL6, IL7, IL13, IL14, and IL 15. In certain embodiments, the interleukin is selected from IL1 β, IL1f9, IL10, IL27, IL33, IL18 BP.

In certain embodiments, wherein the chemokine comprises one or more CC chemokines selected from CCL4, CCL8, CCL6, CCL9, and CCL 11. In certain embodiments, the chemokine comprises one or more CXC chemokines selected from CXCL2, CXCL5, CXCL16, CXCL9 and CXCL 13.

In certain embodiments, wherein the cytokine is selected from the group consisting of IL-1 β, CCL8, and CXCL 5. In certain embodiments, the cytokine comprises IL-1 β and CCL 8. In certain embodiments, the cytokine comprises CCL8 and CXCL 5. In certain embodiments, the cytokines include IL-1 β, CCL8, and CXCL 5.

In certain embodiments, the cell adhesion molecule is selected from ICAM1, CSF3r, Itgam, Siglecf, Adam8, Chl, Sirpa, Nrcam, Emilin2, Emilin1, Tubb6, and/or Parvb.

In certain embodiments, the ADSP source is derived from adipose tissue.

In certain embodiments, the AGC comprises at least one B cell and at least one T follicle helper cell. In certain embodiments, the AGC comprises at least one B cell and at least one dendritic cell. In certain embodiments, the AGC comprises at least one B cell, at least one T follicular helper cell, and at least one dendritic cell. In certain embodiments, the AGC further comprises at least one adipocyte.

In certain embodiments, the AGC comprises PBMCs. In certain embodiments, the PBMCs are isolated from a blood sample derived from human Hematopoietic Stem Cells (HSCs), derived from induced pluripotent stem cells (ipscs), or derived from umbilical cord blood.

In certain embodiments, wherein the antibody-enhancing composition further comprises a co-stimulator, a toll-like receptor (TLR) agonist, a CpG oligodeoxynucleotide (CpG ODN), an anti-apoptotic protein, TNF, Interferon (INF), a lipid, avasimd, EFNB1, EPHB4, a self protein B2, axon-targeting protein 4C, BLIMP-1, IRF4, or any combination thereof.

In certain embodiments, the co-stimulus comprises CD40, CD40L, ICOSL, ICOS, APRIL, B cell activating factor of the TNF family (BAFF), OX40, and/or OX 40L.

In certain embodiments, CpG ODN include CpG2006 and/or D/K CpG.

In certain embodiments, the anti-apoptotic proteins include Bcl-2, Bcl-6, Bcl-XL, Bcl-w, Mcl-1, and/or analogs thereof.

In certain embodiments, the TLR agonist comprises a TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR7/8 agonist, and/or a TLR9 agonist.

In certain embodiments, the method further comprises isolating the antibody from the mixture. In certain embodiments, the method further comprises obtaining a variable region nucleic acid sequence encoding an antibody. In certain embodiments, the method further comprises introducing the nucleic acid sequence into a host cell under conditions suitable for expression of the antibody or antigen-binding fragment thereof. In certain embodiments, the method further comprises assessing whether the antibody specifically binds to the antigen of interest.

In certain embodiments, ADSP is present at a concentration of at least 1ng/ml, 10ng/ml or 50 ng/ml. In certain embodiments, IL2 is present at a concentration of at least 10 ng/ml. In certain embodiments, IL21 is present at a concentration of at least 50 ng/ml.

In certain embodiments, the ADSP is present for at least 1 day. In certain embodiments, IL2 is present for at least 1 day. In certain embodiments, IL21 is present for at least 1 day.

The present disclosure also provides herein a method of inducing proliferation of an antibody-producing cell composition (AGC), B cell activation and differentiation, B cell maturation, and/or promoting class switching in AGC to produce IgG, the method comprising culturing the AGC in a medium comprising IL2, an adipose tissue-derived secreted protein, and/or IL 21. In certain embodiments, the AGC comprises PBMCs. In certain embodiments, the antigen of interest is present in the culture medium.

In certain embodiments, the antibodies produced are fully human monoclonal antibodies.

Also provided herein is a method of producing an antibody or antigen-binding fragment that specifically binds to an antigen of interest, the method comprising:

mixing the antigen, antibody-producing cell composition (AGC) and antibody enhancing composition in a culture medium to form a mixture,

(ii) incubating the mixture in the presence of a nutrient,

wherein the AGC comprises at least one B cell and at least one other type of cell derived from Peripheral Blood Mononuclear Cells (PBMCs), and the antibody enhancing composition comprises IL2, IL21 and one or more adipose tissue-derived secreted proteins (ADSPs).

In certain embodiments, wherein the method further comprises obtaining a variable region nucleic acid molecule encoding an antibody from the mixture. The nucleic acid molecule is optionally introduced into a host cell under conditions suitable for expression of the antibody or antigen-binding fragment thereof. In certain embodiments, wherein the method further comprises isolating the antigen-specific antibody or antigen-binding fragment thereof secreted by the host cell.

The present disclosure also provides herein a composition comprising an isolated antibody-producing cell composition (AGC) comprising at least one B cell and at least one other type of cell derived from Peripheral Blood Mononuclear Cells (PBMCs), the antibody enhancing composition, and a vehicle. In certain embodiments, wherein the composition further comprises an antigen of interest. In certain embodiments, wherein the antibody enhancing composition further comprises IL2 and/or IL 21. In certain embodiments, wherein the AGC comprises at least one B cell and at least one T follicular helper cell. In certain embodiments, wherein the AGC comprises at least one B cell and at least one dendritic cell. In certain embodiments, the AGC comprises at least one B cell, at least one T follicular helper cell, and at least one dendritic cell. In certain embodiments, the AGC comprises PBMCs.

In certain embodiments, the AGC further comprises at least one adipocyte.

In certain embodiments, the antibody-potentiating composition comprises one or more antibody-potentiating factors selected from the group consisting of ADSP, CD40L, ICOSL, ICOS, TLR agonists, and any combination thereof.

The disclosure herein provides a method of identifying an in vitro immune antibody enhancing factor comprising:

a) isolating total RNA from lymph node cells derived from an animal immunized with an antigen of interest;

b) comparing the RNA level of the total RNA isolated in step a) with the RNA level of an unimmunized control animal to determine a gene encoding a protein and having an upregulated expression level;

c) culturing PBMCs in a culture medium comprising the antigen of interest, IL2, IL21 and related proteins;

d) identifying the protein as an antibody enhancer for in vitro immunization if the protein enhances antibody production.

In certain embodiments, the cell is an adipocyte, T follicular helper cell, B cell, or dendritic cell.

In certain embodiments, the protein is expressed by adipocytes, T follicular helper cells, B cells, or dendritic cells.

In one aspect, the present disclosure provides a method of generating a Chimeric Antigen Receptor (CAR), the method comprising expressing a first nucleic acid operably linked to a second nucleic acid, wherein the first nucleic acid encodes an antigen binding domain derived from an antibody or antigen binding fragment thereof produced according to the methods provided herein, and wherein the second nucleic acid encodes a T cell signaling domain.

In one aspect, the present disclosure provides a method of treating cancer in a subject, wherein the method comprises: expressing in a T cell a first nucleic acid operably linked to a second nucleic acid, wherein the first nucleic acid encodes an antigen binding domain derived from an antibody or antigen binding fragment thereof produced according to the methods provided herein, and wherein the second nucleic acid encodes a T cell signaling domain; the T cells are then administered to a subject. In certain embodiments, wherein the T cell is obtained from the subject.

Drawings

FIG. 1 illustrates RNA-seq analysis of up-regulated genes in adipocytes infiltrated into lymph nodes after immunization.

FIG. 2 shows the role of IL-1. beta. in stimulating antibody production.

Figure 3 shows the role of CCL8 in stimulating antibody production.

Figure 4 shows the role of CXCL5 in stimulating antibody production.

Figure 5 shows the effect of CXCL13, CCL4, IL27, CXCL16 and CXCL2 in stimulating antibody production.

FIG. 6 shows that the specificity of antibody production is increased with IL-1. beta. without compromising production efficiency. Fig. 6A and 6B show the levels of anti-OVA IgG and IgM antibody production in the presence or absence of OVA, respectively. FIG. 6C shows the production levels of non-specific anti-BSA IgG antibodies in the presence or absence of OVA.

FIG. 7 shows the expression levels of ADSPsIL-1 β, CCL8, CXCL5, and IL36 in mice with or without OVA immunization.

Detailed Description

The following description of the present disclosure is intended to be illustrative of various embodiments of the disclosure only. Thus, the specific modifications discussed should not be construed as limiting the scope of the disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the disclosure, and it is to be understood that such equivalent embodiments are to be included herein. All references, including publications, patents, and patent applications, cited herein are hereby incorporated by reference in their entirety.

Definition of

As used herein, the term "antibody" includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, multispecific antibody or bispecific (bivalent) antibody or functional portion thereof that binds to a specific antigen. A natural intact antibody comprises two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. Each heavy chain consists of a variable region (VH) and first, second and third constant regions (CH 1, CH2 and CH3, respectively), while each light chain consists of a variable region (VL) and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ, and μ, and mammalian light chains as λ or κ. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains are usually subdivided into three hypervariable regions, called Complementarity Determining Regions (CDRs) (light chain (L) CDRs, including LCDR1, LCDR2 and LCDR 3; heavy chain (H) CDRs, including HCDR1, HCDR2, HCDR 3). CDR boundaries of the antibodies and antigen-binding fragments disclosed herein can be defined or identified by conventions including Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A.M., J.mol.biol.,273(4),927(1997), Chothia, C. et Al, J Mol biol.Dec 5; 186(3):651-63 (1985)), Chothia, C.and Lesk, A.M., J.mol.biol.,196,901(1987), Chothia, C. et Al, Nature.Dec 21-28; 342(6252):877-83(1989), Kabat E.A. et Al, National instruments of Healthh, Bethesda, Md (1991)). These three CDRs are inserted between flanking sequences called Framework Regions (FRs), which are more conserved than the CDRs and form a scaffold that supports hypervariable loops. Thus, each VH and VL comprises, in order, three CDRs and four FRs (amino acid residues N-terminal to C-terminal): FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are classified into five main classes according to the amino acid sequence of the antibody heavy chain constant region: IgA, IgD, IgE, IgG and IgM, characterized by the presence of α, δ, ε, γ and μ heavy chains. Subclasses of several major antibody classes include IgG1(γ 1 heavy chain), IgG2(γ 2 heavy chain), IgG3(γ 3 heavy chain), IgG4(γ 4 heavy chain), IgA1(α 1 heavy chain), or IgA2(α 2 heavy chain).

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., each antibody comprising the population is identical and/or binds the same epitope, except for possible variant antibodies. Contain natural mutations or mutants produced during monoclonal antibody production, such variants usually being present in small amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention can be prepared by a variety of techniques, including but not limited to hybridoma methods, recombinant DNA methods, phage display methods.

As used herein, with respect to an antibody or antigen-binding fragment, the term "fully human antibody" refers to a product of the antibody or antigen-binding fragment having an amino acid sequence corresponding to or consisting of the amino acid sequence of the antibody produced by human or human immune cells, or derived from a non-human source, such as a transgenic non-human animal utilizing a human antibody repertoire or other human antibody coding sequences. In certain embodiments, a fully human antibody does not comprise amino acid residues (particularly antigen binding residues) derived from a non-human antibody.

An "affinity matured" antibody is one that has alterations or substitutions of one or more amino acid residues in one or more hypervariable regions (HVRs), such as Complementarity Determining Regions (CDRs), which increase the affinity of the antibody for an antigen compared to a parent antibody without such alterations or substitutions.

As used herein, the term "antigen-binding fragment" refers to a fragment formed from an antibody fragment that comprises one or more CDRs, or any other antibody portion that binds an antigen but does not comprise the entire native antibody structure. In certain embodiments, the antibodies provided herein are antigen binding fragments. Examples of antigen binding fragments include, but are not limited to, diabodies, Fab ', F (ab')₂Fv fragment, disulfide-stabilized Fv fragment (dsFv), (dsFv)₂Bispecific antibodies dsFv (dsFv-dsFv'), disulfide stabilized diabodies (ds diabodies), single chain antibody molecules (scFv), scFv dimers (diabodies), multispecific antibodies, camel single domain antibodies, nanobodies, domain antibodies, isolated CDRs, and diabodies. The antigen binding fragment is capable of binding to the same antigen to which the parent antibody binds. In certain embodiments, the antigen-binding fragment may comprise one or more CDRs from a particular human antibody.

As used herein, "antigen" or "Ag" refers to a compound, composition, peptide, polypeptide, protein, RNA, DNA, bacterial virus, or any immunogenic substance that can stimulate antibody production, or a T cell response in cell culture or in an animal, including a composition added to cell culture (e.g., a hybridoma), or a composition injected or absorbed into an animal. The antigen reacts with a particular humoral or cellular immune product (e.g., an antibody), including a heterologous antigen-induced reaction.

"Fab" in reference to an antibody refers to a monovalent antigen-binding fragment of an antibody consisting of a single chain light chain (variable and constant regions) bound by disulfide bonds to the variable and first constant regions of a single chain heavy lotus. Fab may be obtained by papain digestion of the N-terminal residues of the disulfide bonds between the heavy chains of the antibody hinge region.

"Fab'" refers to a Fab fragment that includes a portion of the hinge region, which can be obtained by pepsin digestion of the C-terminal residues of the disulfide bonds between the heavy chains of an antibody. Thus, the few residues in the hinge region (including one or more cysteines) are different from Fab.

“F(ab')₂"refers to a dimer of Fab' comprising two light chains and a portion of two heavy chains.

By "Fc" of an antibody is meant the portion of the antibody consisting of the second and third constant regions of the first heavy chain bound to the second and third constant regions of the second heavy chain by disulfide bonds. The IgG and IgM Fc regions comprise three heavy chain constant regions (the second, third and fourth heavy chain constant regions in each chain). It can be obtained by papain digestion of antibodies. The Fc portion of an antibody is responsible for various effector functions, such as ADCC and CDC, but does not function in antigen binding.

"Fv" with respect to an antibody refers to the smallest fragment of an antibody that carries an intact antigen binding site. The Fv fragment consists of a single heavy chain variable region joined to a single light chain variable region. "dsFv" refers to a disulfide-stabilized Fv fragment in which the variable regions of individual light chains and the variable regions of individual heavy chains are linked by disulfide bonds.

"Single chain Fv antibody" or "scFv" refers to an engineered antibody consisting of a light chain variable region and a heavy chain variable region joined to each other either directly or through a peptide linker sequence (Huston JS et al Proc Natl Acad Sci USA,85:5879 (1988)). "scFv dimer" refers to a single chain comprising two heavy chain variable regions and two light chain variable regions and a linker. In certain embodiments, an "scFv dimer" is a bivalent diabody or bivalent scFv (bsfv) comprising a VH-VL that is dimerized with another VH-VL moiety (connected by a peptide linker) such that the VH of one moiety coordinates to the VL of the other. And form two binding sites that can target the same antigen (or peptide) or different antigens (or peptides). In other embodiments, an "scFv dimer" is a bispecific diabody comprising VH1-VL2 (linked by a peptide linker) associated with VL1-VH2 (also linked by a peptide linker) such that VH1 and VL1 coordinate and VH2 and VL2 coordinate and each pair has a different antigen specificity.

"Single chain Fv-Fc antibody" or "scFv-Fc" refers to an engineered antibody consisting of an scFv linked to the Fc region of an antibody.

"Camel single domain antibody", "heavy chain antibody", "nanobody" or "HCAb" refers to an antibody comprising two VH domains and no light chain (Riechmann L. and Muydermans S., J Immunol methods. Dec10; 231(1-2):25-38 (1999); Muydermans S., J Biotechnol. Jun; 74(4):277-302 (2001); WO 94/04678; WO 94/25591; U.S. patent No.6,005,079). Heavy chain antibodies originally came from the camelidae family (camel, dromedary, and llama). Camel antibodies have authentic antigen binding epitopes, although no light chain is present (Hamers-Casterman C. et al, Nature. Jun 3; 363(6428):446-8 (1993); Nguyen VK. et al, "Heavy-chain antibodies in Camellia; a case of evolution innovation," immunogenetics. Apr; 54(1):39-47 (2002); Nguyen VK. et al immunology. May; 109(1):93-101 (2003)). The variable domain of the heavy chain antibody (VHH domain) represents the smallest known antigen-binding unit produced by an adaptive immune response (Koch-Nolte F. et al, FASEB J. Nov; 21(13):3490-8.Epub 2007Jun 15 (2007)). "diabodies" include small antibody fragments with two antigen-binding sites, where the fragment comprises a VH domain (VH-VL or VL-VH) linked to a VL domain in a single polypeptide chain (see, e.g., Holliger P. et al, Proc Natl Acad Sci U S A. Jul 15; 90(14):6444-8 (1993); EP 404097; WO 93/11161). Because the linker is too short, the two domains on the same chain cannot pair, and therefore, these domains are forced to pair with the complementary domains of the other chain, thereby creating two antigen binding sites. The antigen binding sites may target the same site of different antigens (or epitopes).

"Domain antibody" refers to an antibody fragment comprising only heavy chain variable regions or light chain variable regions. In certain embodiments, two or more VH domains are covalently linked to a peptide linker to form a bivalent or multivalent domain antibody. The two VH domains of a bivalent domain antibody may target the same or different antigens.

In certain embodiments, "(dsFv)₂"comprises three peptide chains: the two VH moieties are linked by a peptide linker and a disulfide bond is associated with the two VL moieties. In the case of a high degree of similarity between two numerical values, one skilled in the art will not recognize or consider that there is a significant difference between the two numerical values, or that the difference is small in terms of the statistical and/or biological activity indicated by the numerical values. Conversely, "substantially lower" means a value of less than about 50%, less than about 40%, less than about 30%, less than about 20%, less than about 10% as a function of the reference value.

As used herein, the term "specific binding" or "specifically binds" refers to a non-random binding reaction between two molecules, e.g., between an antibody and an antigen. In certain embodiments, an antibody or antigen-binding fragment provided herein specifically binds a human and/or non-human antigen with a binding affinity (KD) of about 0.01nM to about 100nM, about 0.1nM to about 100nM, 0.01nM to about 10nM, about 0.1nM to about 10nM, about 0.01nM to about 5nM, about 0.1nM to about 5nM, about 0.01nM to about 1nM, about 0.1nM to about 1nM, or about 0.01nM to about 0.1 nM. As used herein, K_DRefers to the ratio of the dissociation rate to the association rate (k)_off/k_on) It may be determined using a surface plasmon resonance method, for example using an instrument such as Biacore.

As used herein, "treating," "treatment," or "therapy" of a condition may be used interchangeably and includes therapeutic treatment, prophylactic or defensive measures, such as preventing or alleviating the condition, slowing the onset or rate of progression of the condition, reducing the risk of developing the condition, preventing or delaying the progression of the condition associated with the condition, alleviating or ending the condition associated with the condition, causing the condition to resolve, cure, or some combination thereof.

As used herein, the term "vector" refers to a vehicle into which a polynucleotide encoding a protein is operably inserted and transported so that the protein is expressed in a host cell. The vector may be used to transform, transduce or transfect a host cell so that it expresses a genetic element carried within the host cell. Exemplary types of vectors include, but are not limited to, plasmids (e.g., phagemids, cosmids, Yeast Artificial Chromosomes (YACs), Bacterial Artificial Chromosomes (BACs) or P1-derived artificial chromosomes (PACs)), viral vectors (bacteriophages such as lambda phage or M13 phage or animal viruses), bacterial vectors or non-epidemic mammalian vectors. Classes of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-chaperones, herpes viruses (e.g., herpes simplex), poxviruses, baculoviruses, papilloma viruses, and papilloma viruses (e.g., SV 40). The vector may contain a variety of elements for controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector (e.g., a bacterial vector or an episomal mammalian vector) can comprise an origin of replication. The vector may also include materials that facilitate its entry into the cell, including but not limited to viral particles, liposomes, or protein capsids.

As used interchangeably herein, "nucleic acid" or "nucleic acid sequence" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses polynucleotides containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al, Nucleic Acid Res.19:5081 (1991); 0htsuka et al, J.biol.chem.260: 2605-.

The term "host cell" as used herein refers to a cell into which an exogenous polynucleotide and/or vector has been introduced to express one or more exogenous proteins. The cell refers to a cell of a specific subject and its progeny. The host cell may be a prokaryote, eukaryote, plant cell, animal cell, or hybridoma. The cell may be one that does not express the protein at the desired level but contains the nucleic acid thereof unless a modulator is introduced into the cell or a regulatory sequence is introduced into the host cell such that it is operably linked to the nucleic acid.

As used herein, the term "animal" refers to a mammal, such as a human, camelid, mouse, rat, rabbit, goat, sheep, guinea pig, or hamster. In certain embodiments, the animal is a human.

The "isolated" material has been artificially altered from a natural state. If an "isolated" component or substance occurs in nature, it is said to have been altered or deleted from its original environment, or both. For example, an "isolated" polynucleotide or polypeptide is a polynucleotide or polypeptide that is free of other polynucleotides or polypeptides, respectively, and is independent of the natural components with which the polynucleotide or polypeptide is associated in its natural state. In certain embodiments, an "isolated" protein is purified by at least one step to a purity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, as determined by electrophoretic methods (e.g., SDS-PAGE using coomassie blue or silver staining, isoelectric focusing, capillary electrophoresis), chromatography (e.g., ion exchange chromatography or reverse phase HPLC), or Lowry methods.

Activation-induced cytidine deaminase, also known as AICDA and AID, is a 24kDa enzyme that is encoded by the AICDA gene in humans. ATP is a member of the cytidine deaminase family, which is involved in somatic hypermutation and class switch recombination of immunoglobulin genes in B cells, and is considered to be a major regulator of secondary antibody diversification. AID induces DNA mutations that change cytosine to uracil (identified as thymine during DNA replication), which convert C: g is converted into T: a or A: and a T base pair. During somatic hypermutation, antibodies are mutated to generate libraries of antibody variants with various affinities.

PR domain zinc finger protein 1, also known as blip-1, is a transcriptional repressor protein encoded by the PRDM1 gene in humans. BLIMP-1 specifically binds to PRDI (positive regulatory domain I element) of the beta-interferon (beta-IFN) gene promoter and suppresses the gene expression of beta-IFN. The increase of blip-1 protein in B lymphocytes, T lymphocytes, NK cells and other immune cells leads to an immune response by promoting proliferation and differentiation of antibody-secreting plasma cells.

The present disclosure provides a method for in vitro immunization, in vitro induction of a humoral response, i.e., in vitro production of antigen-specific human antibodies, due to antigen recognition by immunoglobulins expressed on the surface of native human B lymphocytes cultured in vitro with the antigen.

In animal immunization, Germinal Centers (GCs) are important sites in lymph nodes and spleen where mature B cells proliferate, differentiate and mutate their antibody genes by somatic hypermutation to achieve higher affinity and switch the antibody class from IgM to IgG during the immune response. As a center for the production of affinity matured B cells and persistent memory B cells, GC is important in the humoral immune response of B cells. In GC, B cells undergo rapid and mutant cell division in the dark region (referred to herein as central cells) and migrate to the light region (referred to herein as central cells), where follicular dendritic cells assist in their selection by follicular helper T cells. Those selected B cells returned to the dark area for further division and mutation. At the same time, a small number of memory B cells and plasma cells leave the GC.

Unlike GC in animals, in vitro immunization methods are capable of producing antibodies in GC-like B cells in vitro in Peripheral Blood Mononuclear Cells (PBMCs).

Antibody-producing cell compositions (AGC)

The term "antibody-producing cell composition" or "AGC" refers to a group of cells that produce an antigen-specific antibody of interest under suitable conditions for antibody production. In certain embodiments, the AGC comprises at least one B cell and at least one other type of cell derived from Peripheral Blood Mononuclear Cells (PBMCs). In certain embodiments, the AGC comprises PBMCs.

Peripheral Blood Mononuclear Cells (PBMC) are any peripheral blood cells with a circular nucleus, including lymphocytes and monocytes. PBMCs can be extracted from whole blood by techniques conventional in the art, such as density gradient centrifugation (a hydrophilic polysaccharide that can separate the blood layer) using ficoll, separating the blood into top plasma, middle PBMCs and bottom polymorphonuclear cells (e.g., neutrophils and eosinophils) and erythrocytes. Proliferation of PBMCs can be detected or confirmed in vitro by methods known in the art, for example, by MTT assay (colorimetric), AO/PI (acridine orange and propidium iodide) staining or cell counting. In certain embodiments, the PBMCs are isolated from a whole blood sample. In certain embodiments, the PBMCs are derived from human Hematopoietic Stem Cells (HSCs), induced pluripotent stem cells (ipscs) or cord blood.

In certain embodiments, the AGC is an isolated cell-like mixture comprising lymphocytes (T cells, B cells, NK cells), monocytes, macrophages, and dendritic cells.

In certain embodiments, the AGC comprises PBMCs. In certain embodiments, the AGC comprises at least one B cell. In certain embodiments, the AGC comprises at least one of at least one B cell, at least one T cell (e.g., T follicular helper cell), at least one dendritic cell, at least one NK cell, at least one monocyte, and at least one adipocyte.

For example, in certain embodiments, the AGC comprises at least one B cell and at least one T cell (e.g., T follicular helper cell). In certain embodiments, the AGC comprises at least one B cell and at least one dendritic cell. In certain embodiments, the AGC comprises at least one B cell, at least one T cell (e.g., T follicular helper cell), and at least one dendritic cell. In certain embodiments, the AGC comprises at least one B cell and at least one NK cell. In certain embodiments, the AGC comprises at least one B cell and at least one monocyte. In certain embodiments, the AGC comprises at least one B cell, T cell (e.g., T follicular helper cell), and at least one NK cell. In certain embodiments, the AGC comprises at least one B cell, at least one T cell (e.g., T follicular helper cell), at least one dendritic cell, and at least one NK cell.

In certain embodiments, the AGC comprises at least one adipocyte and at least one B cell. In certain embodiments, the AGC comprises at least one adipocyte, at least one B cell, and at least one T cell (e.g., T follicular helper cell). In certain embodiments, the AGC comprises at least one adipocyte, at least one B cell, and at least one dendritic cell. In certain embodiments, the AGC comprises at least one adipocyte, at least one B cell, at least one T cell (e.g., T follicular helper cell), and at least one dendritic cell.

In certain embodiments, at least one of the B cells, T follicular helper cells, dendritic cells and adipocytes is a human cell. In certain embodiments, the B cell is a human B cell. In certain embodiments, the PBMCs are derived from human PBMCs.

In certain embodiments, the PBMCs are isolated from a human donor. In certain embodiments, the PBMCs are derived from stem cells.

As used herein, the term "B cell" refers to a B lymphocyte, a lymphocyte subtype of a leukocyte. They play a role in the humoral immunity of the adaptive immune system by secreting antibodies. B cells also present antigen and secrete cytokines. In mammals, B cells mature in the bone marrow. After B cells mature in the bone marrow, they migrate through the blood to Secondary Lymphoid Organs (SLOs), such as the spleen and lymph nodes, where they continuously receive antigen via circulating lymph fluid. Unlike the other two types of lymphocytes, i.e., T cells and natural killer cells, B cells express a B Cell Receptor (BCR) on their cell membrane, thereby allowing the B cell to bind to a specific antigen, thereby initiating an antibody response against the antigen. In these three B cell subsets, FO B cells preferentially undergo T cell dependent (TD) activation, while Marginal Zone (MZ) B cells and B1B cells preferentially undergo T cell independent (TI) activation. TI antigen-activated B cells diffuse out of the lymphoid follicles but, still in SLOs, may undergo immunoglobulin class switching and differentiate into short-lived plasma cells, producing early weak antibodies, mainly of the IgM class, along with some long-lived non-proliferating antibody-producing plasma cells. The enhancement of B cell activity is mediated by activation of CD21, CD21 is a surface receptor, forming a complex with the surface proteins CD19 and CD81 (collectively the three are referred to as the B cell co-receptor complex, BCR). When BCR binds to an antigen labeled with a fragment of the C3 complement protein, CD21 binds to the C3 fragment, co-ligates with the bound BCR, and signals through CD19 and CD81 to lower the activation threshold of the cell.

In certain embodiments, the B cells are those naturally occurring in PBMCs from healthy donors. In certain embodiments, the B cell is a cultured B cell, a B cell differentiated from a stem cell or a B cell isolated from an animal. In certain embodiments, the B cell is a B cell genetically engineered to produce a non-naturally occurring antibody, e.g., a bispecific antibody.

The term "native B lymphocyte" refers to a B lymphocyte (B cell) that has never encountered an antigen that can be bound by the paratope expressed by its surface immunoglobulin. These B cells are directly from the peripheral blood of a subject who has not been contacted with the antigen. Thus, these subjects will exhibit a seronegative status with respect to the antigen, i.e. they will exhibit a serum antibody titer specific for the antigen that is not detectable.

The term "T cell" as used herein refers to a lymphocyte derived from the thymus and primarily involved in cellular immunity. Examples of T cells include CD4+ T cells (T helper, TH cells), CD8+ T cells (cytotoxic T cells, CTLs), memory T cells, regulatory T cells (Treg cells, e.g., activated tregs and unactivated tregs), apoptotic T cells, naive T cells, or other T cell populations.

In certain embodiments, the T cells are those naturally occurring in PBMCs from healthy donors. In certain embodiments, the T cell is a cultured T cell, a T cell differentiated from a stem cell or a T cell isolated from an animal. In certain embodiments, the T cell is a genetically engineered T cell.

A "T helper cell" is a T cell type that participates in the adaptive (i.e., tailored to a particular pathogen) immune system by releasing T cell cytokines, thereby suppressing or modulating the immune response. T helper cells are involved in the switch of B cell antibody classes, activation and growth of cytotoxic T cells, and in maximizing the bactericidal activity of phagocytic cells (e.g., macrophages). Mature T helper cells are CD4 positive and are activated by cytokine release and cell-cell interactions (e.g., CD40 (on APC) and CD40L (on T follicle helper cells)). T helper cells can develop into two major subtypes, Th1 and Th2 cells. Th1 helper cells are involved in the cellular immune system against intracellular bacteria and protozoa and are triggered by IL-12 and release IFN-. gamma.and IL-2. Th1 helper cells help to enhance killing of macrophages, proliferation of CD8+ T cells, production of B cell IgG, and secretion of IFN- γ into CD4+ T cells. Th2 helper cells are involved in the humoral immune system against extracellular parasites and are triggered by IL-4 and IL-2 and release IL-4, IL-5, IL-9, IL-10, IL-13 and IL-25. Th2 helper cells help eosinophils, basophils, mast cells, stimulate B cell proliferation and antibody production, and IL-4/IL-5 secreting CD4+ T cells. T follicular helper cells are located at the periphery of the B cell follicles (e.g. lymph nodes, spleen and Peyer's patches) of secondary lymphoid organs and are identified by the constitutive expression of the B cell follicle homing receptor CXCR 5. TFH cells trigger the formation and maintenance of germinal centers by expression of CD40L and expression of CD40L and secretion of IL-21 and IL-4 upon interaction and cross-signaling with their cognate follicular (Fo B) B cells.

The term "antigen presenting cell" or APC is intended to mean a cell expressing one or more Major Histocompatibility Complex (MHC) class I and class II molecules (HLA class I and class II molecules in humans), as well as a cell capable of presenting an antigen to CD4+ T and CD8+ T lymphocytes specific for that antigen. As antigen presenting cells, mention may in particular be made of Dendritic Cells (DCs), Peripheral Blood Mononuclear Cells (PBMCs), monocytes, macrophages, B lymphocytes, lymphoblastoid cell lines and human or animal cell lines modified by the expression of class I and class I MHC molecules, in particular HLA I and HLA II molecules and the like.

The terms "cytotoxic T cell", "T-killer cell" or "CTL" as used herein are interchangeable and refer to a type of T cell that recognizes a particular antigen produced by a cancer cell, a virus-infected cell or otherwise damaged cell. Antigen is brought to the cell surface by MHC class I, and with the help of CD8, the TCR binds to the TCR on cytotoxic T cells. Thus, cytotoxic T cells were CD8 positive.

Memory T cells are a subset of T cells that have previously undergone (encountered and responded to) cancer cells, bacterial or viral antigens. The memory T cells may be CD4+ and/or CD8+ T cells, or memory cytotoxic T cells. After re-exposure to antigen, long-lived memory T cells can mediate a faster, more efficient secondary response. This memory function may be provided by CD4+ and/or CD8+ memory T cells. Long-acting memory T cells differ from effector cells, which have a short lifespan and usually die following an immune response by activation-induced cell death (AICD). However, between the two cell types, transitional forms exist, such as effector memory cells. Like effector cells, they are able to patrol the whole body and exert effector functions upon antigen exposure, and they can proliferate and have a longer life than effector cells.

As used herein, "regulatory T cells" or "Tregs" refer to subpopulations of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune responses. Tregs have immunosuppressive effects and participate in the suppression of autoreactive immune responses. Tregs are positive for CD4, CLTA4, GITR, neuroxilin-1 and CD 25. Tregs perform their suppressive function on activated T cells through contact-dependent mechanisms and cytokine production (Fehervari, Z. & Sakaguchi, Curr Opin Immunol 16,203-8 (2004)). Tregs also modulate immune responses by interacting directly with ligands on Dendritic Cells (DCs), e.g., CTLA4 interacts with the B7 molecule on DCs, thereby inducing the production of indoleamine 2, 3-dioxygenase (IDO) (falllarino, f. et al, Nat Immunol 4,1206-12(2003)), and CD40L linkages (Serra, p. et al, immunonity 19,877-89 (2003)).

As used herein, "Natural Killer (NK) cell" refers to a lymphocyte that normally expresses a cell surface marker, but does not express CD16 and/or NCAM and/or CD56 molecules of CD 3. NK cells refer to cells that exist in purified cell populations in vivo or in vitro in mammals. NK cells are a cytotoxic lymphocyte that is critical to the innate immune system. NK cells act like cytotoxic T cells.

"Dendritic Cells (DCs)" are potent Antigen Presenting Cells (APCs) that process antigenic material and present it to T cells on the cell surface. After activation, DCs migrate to lymph nodes where they interact with T and B cells, initiating and modeling adaptive immune responses. Human dendritic cells selectively express CD 83. DCs have a variety of surface receptors that can be used to recognize a variety of pathogens. In addition, DCs are able to sense various endogenous messengers, such as cytokines and chemokines, as well as surface molecules on other cells of the immune system. DCs process various input signals through intracellular signaling pathways, thereby triggering various differentiation programs. Dendritic cells are capable of inducing primary T cell responses in vitro and in vivo. In vitro, DCs can be loaded with various protein and peptide antigens as well as tumor cell extracts (Nestle, F. et al, nat. Med.,4:328-332 (1998)). DC cells can also be transduced by genetic means to express these tumor antigens. For immunization purposes, DCs can also be fused directly to tumor cells (Kugler, A. et al, nat. Med.,6: 332-.

In certain embodiments, the DCs are DCs that occur naturally in PBMCs from healthy donors. In certain embodiments, the DC is a cultured DC, a differentiated DC from a stem cell or a DC isolated from an animal. In certain embodiments, the T cell is a genetically engineered form of a DC.

Adipocytes refer to lipocytes and adipocytes, which are cells that mainly constitute adipose tissue. After immunization of animals, it was found that lymph peripheral fat cells were tightly bound to lymph nodes and even infiltrated into lymph nodes. Secreted proteins derived from adipose tissue (ADSP) refer to proteins that are capable of enhancing AGC production provided herein to antibodies, secreted within or by adipose tissue associated with lymph nodes. When an animal is antigen-immunized, if its protein level or RNA level is up-regulated by at least two-fold, ADSP can be recognized, compared to the same animal before antigen-immunization or an animal not antigen-immunized. Although ADSP is secreted by adipocytes, it can also be produced and secreted by other cell types derived from Peripheral Blood Mononuclear Cells (PBMC), such as B cells, T cells (e.g., T follicular helper cells), or dendritic cells.

In certain embodiments, the adipocytes are cultured adipocytes, adipocytes differentiated from stem cells, or adipocytes isolated from an animal.

At least one type of monocyte, such as a B cell, a T cell (e.g., a T follicle helper cell), a dendritic cell, an NK cell, a monocyte, a Hematopoietic Stem Cell (HSC), bone marrow, neonatal umbilical cord blood (thus known as Cord Blood Mononuclear Cell (CBMC)), amniotic fluid or a pluripotent stem cell (hPSC), including Embryonic Stem Cells (ESC) and Induced Pluripotent Stem Cells (iPSC)) can be isolated and/or reconstituted from whole blood of a subject. In certain embodiments, wherein the at least one type of monocyte may be from an adult, adolescent, or child.

Hematopoietic Stem Cells (HSCs) are located in the red bone marrow and produce various types of mature blood cells during hematopoiesis, including myeloid cells (monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells and megakaryocytes) or platelets) and lymphoid cells (T cells, B cells and natural killer cells). Bone marrow is a spongy or spongy semi-solid tissue in the bone, consisting of hematopoietic cells (bone marrow and lymphoid lineage), bone marrow adipose tissue, Mesenchymal Stem Cells (MSCs), and supporting stromal cells. Human bone marrow typically produces about 5000 million blood cells per day that enter the circulation through a permeable vascular sine wave within the marrow cavity. Lymphoid cells mature in other lymphoid organs such as the thymus.

Cord blood includes a number of immunologically immature neonatal cord blood mononuclear cells (UCBMC) and has also been reported as a source of hematopoietic stem cells (see Gluckman E et al, an HLA homozygote cord blood hematopoietic reconstitution for Fanconi anemic patients N Engl J Med.1989Oct 26; 321(17): 1174-8.). In vitro, monocytes and/or HSCs can be distinguished from Human Pluripotent Stem Cells (hPSCs), including Human Embryonic Stem Cells (ESCs) and Induced Pluripotent Stem Cells (iPSCs), such as primitive blood endothelial cell precursors, mature myeloid Cells, Cells of the erythroid and lymphoid lineages (Melinda K.Hexum et al, in vivo evaluation of Human Pluripotent Stem cell hematopoietic Stem Cells, Human Pluripotent Stem Cells,2011. 433. 447. amniotic fluid also contains monocytes and Cells with hematopoietic activity (see Ditadi A et al, Human and mouse amniotic fluid c-Kit + Lin Cells show hematopoietic activity, blood.2009Apr 23; 113(17): 3953-60).

The cells provided herein for producing an antibody or antigen-binding fragment thereof can be cultured in a variety of media. Commercially available media such as Ham's F10(Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing the antibody-producing cell composition (AGC). In addition, Ham et al, meth.Enz.58:44(1979), Barnes et al, anal. biochem.102:255(1980), U.S. Pat. No.4,767, 704; 4,657,866, respectively; 4,927,762, respectively; 4,560,655, respectively; or 5,122,469; WO 90/03430; WO 87/00195; or any of the media described in U.S. Pat. No. re.30,985 may be used as the medium for the antibody-producing cell composition (AGC). Any of these media may be supplemented as needed with hormones and/or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCINTM drugs), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be added at appropriate concentrations known to those skilled in the art. It will be apparent to one of ordinary skill that culture conditions, e.g., temperature, pH, etc., are those previously used for antibody-producing cell compositions (AGC).

Antibody enhancing compositions

As used herein, the term "antibody enhancing composition" refers to a collection of factors capable of enhancing the amount of antibody produced in an in vitro antibody producing system. Under in vitro immune conditions, the antibody-enhancing composition is capable of inducing proliferation, B cell activation and differentiation, B cell maturation, T cell differentiation, antibody affinity maturation, antibody diversity and/or promoting class switching in antibody-producing PBMCs to produce IgG. In certain embodiments, the antibody-enhancing composition comprises one or more antibody-enhancing factors.

In certain embodiments, the antibody enhancing composition or antibody enhancing factor is selected from adipose tissue-derived secreted protein (ADSP), compounds that interact with CD40 and CD40L, ICOS-and ICOS-L-interacting compounds, TLR agonists, OX40, OX40L, APRIL (proliferation-inducing ligand), BAFF, CR2, chemokines (CXCL5, CXCL16, CXCL9, CXCL12(SDF-1), CXCL13, CXCL16), Flt-3L, interleukins (IL1(α/β), IL2, IL3, IL4, IL5, IL6, IL7, IL10, IL13, IL14, IL15, IL21, IL27, IL33, IL1f9, IL18 IMD 18BP), SAP (signal lymphocyte activating molecule [ SLAM ] associated protein), staphylococcal A inactivated Cowan (SAC 1), heat-fixed lipoprotein (SAC 848), and other proteins such as SAC-immobilized lipoprotein (SAC-LPS), and HAIR 848 (SAC-LPS) TSLP, Tumor Necrosis Factor (TNF) α, type I interferons (e.g., IFN α/β), type II interferons (e.g., IFN γ), lipids, avasimid, EFNB1, EPHB4(Lu et al, Science, 2017, eaai9264), self protein B2, axon-guided protein 4C (Hu et al, CellReports, 2017, 19, 995-.

In certain embodiments, the antibody-enhancing composition or antibody-enhancing factor is selected from adipose tissue-derived secreted protein (ADSP), CD40L, ICOSL, ICOS, TLR agonists, and any combination thereof.

In certain embodiments, the adipose tissue-derived secreted protein enhances antibody production by AGC, B cell activation and differentiation in AGC, and/or B cell maturation in AGC.

In certain embodiments, the ADSP level or RNA level thereof in the immunized animal is 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 15-, 20-, 25-, 30-, 35-, 40-, 45-, or 50-fold higher than that of an unimmunized control animal or the same animal prior to immunization.

Antibody enhancers are intended to encompass any form, e.g., 1) naturally unprocessed molecules, naturally occurring variants of "full-length" molecular chains or molecules, including, for example, splice variants or allelic variants; 2) processing any form produced in the cell; or 3) full-length fragments (e.g., truncated forms, antibody enhancing domains); modified forms produced by recombinant methods (e.g., mutant forms, glycosylated/pegylated, His-tag/immunofluorescence fusion forms); or 4) homologues of other species.

In certain embodiments, the ADSPs comprise cytokines and cell adhesion molecules capable of enhancing antibody production. In certain embodiments, the ADSP is capable of increasing the percentage of IgG in total antibody production.

The cytokine is a small protein

Are important in cell signaling, including autocrine signaling, paracrine signaling, and endocrine signaling as immunomodulators. Cytokines can be produced by immune cells, such as macrophages, B lymphocytes, T lymphocytes, and mast cells, as well as endothelial cells, fibroblasts, and various stromal cells. Cytokines include chemokines, interferons, interleukins, and Tumor Necrosis Factor (TNF).

Interferons (IFNs) belong to a class of proteins called cytokines, molecules used for communication between cells to trigger the protective defense system of the immune system, contributing to the elimination of pathogens. IFNs are a group of signaling proteins that are produced and released by host cells in response to the presence of a variety of pathogens (e.g., viruses, bacteria, parasites, and tumor cells). Interferons also have a variety of other functions: they can activate immune cells such as natural killer cells and macrophages. They up-regulate antigen presentation by increasing expression of Major Histocompatibility Complex (MHC) antigens, thereby enhancing host defense. IFNs are generally classified into three categories: type I IFN, type II IFN and type III IFN.

The Tumor Necrosis Factor (TNF) superfamily is a superfamily of type II transmembrane proteins that contain TNF homology domains and form trimers. Members of this superfamily are released from cell membranes by extracellular proteolytic cleavage and function as cytokines. These proteins are expressed primarily by immune cells and regulate a variety of cellular functions, including regulation of immune responses and inflammation, as well as regulation of proliferation, differentiation, apoptosis, and embryogenesis.

Interleukin (IL) is a cytokine that is first expressed by leukocytes with complex immunoregulatory functions including cell proliferation, maturation, migration and adhesion, immune cell differentiation and activation, and inflammatory and anti-inflammatory effects. A minority of the members act as chemokines for helper T cells, in parallel with the role of chemokines. Other viruses are closely related to the response of cells to viral pathogens, making them similar to IFNs. IL is a very important mediator in the management of the physiological response to infection and also plays an important role in the pathophysiology of a variety of diseases.

In certain embodiments, the cytokine is an interleukin. In certain embodiments, the interleukin is selected from IL1 β, IL1f9, IL10, IL27IL33, and IL18 BP.

As used herein, the term "IL 10" refers to interleukin 10, an anti-inflammatory cytokine, also known as human Cytokine Synthesis Inhibitory Factor (CSIF). IL10 signals through a receptor complex consisting of two IL10 receptor 1 and two IL10 receptor 2 proteins. An exemplary complete cDNA sequence for human IL10 has accession number AY029171.1, and an exemplary amino acid sequence for human IL10 has accession number AAK 38162.1. The term "IL 10" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants.

As used herein, the term "IL 1 β" refers to one of the interleukin-1 β in the interleukin-1 family, which is a monokine produced by monocytes and macrophages and corresponds to an inflammatory cytokine. It is a potent proinflammatory cytokine. It was originally found to be a major endogenous pyrogen that induces prostaglandin synthesis, neutrophil influx and activation, T cell activation and cytokine production, B cell activation and antibody production as well as fibroblast proliferation and collagen production. Promoting the differentiation of Th17 of T cells. In synergy with IL 12/interleukin 12, T helper 1(Th1) cells were induced to synthesize IFNG (Tominaga K. et al, int. Immunol.2000,12: 151-160). The GENBANK accession number for an exemplary complete cDNA sequence of human IL1 β is BC008678.1 and the GENBANK accession number for an exemplary amino acid sequence of human IL1 β is AAH 08678.1. The term "IL 1 β" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "IL 1F 9" refers to interleukin-1 family member 9, also known as interleukin 36 γ. It is a member of the interleukin 1 cytokine family. The activity of this cytokine is mediated by interleukin-1 receptor-like 2(IL1RL2/IL1R-rp2/IL-36 receptor) and is specifically inhibited by interleukin-36 receptor antagonists (IL36RA/IL1F5/IL1 delta). The expression of this cytokine in keratinocytes can also be induced by various pathogen-associated molecular patterns (PAMPs). The GENBANK accession number for an exemplary complete cDNA sequence of human IL1F9 is BC096721.1 and the GENBANK accession number for an exemplary amino acid sequence of human IL1F9 is AAH 96721.1. The term "IL 1F 9" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants.

The term "IL 27" as used herein refers to interleukin 27, a member of the IL-12 cytokine family. IL27 has pro-inflammatory and anti-inflammatory properties, regulates T helper cell development, inhibits T cell proliferation, stimulates cytotoxic T cell activity, induces B cell isotype switching, and has multiple effects on innate immune cells. An exemplary complete cDNA sequence for human IL27 has accession number BC062422.1 and an exemplary amino acid sequence for human IL27 has accession number GENBANK of AAH 62422.1. The term "IL 27" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "IL 33" refers to the member interleukin 33 of the IL-1 family, which effectively drives the production of cytokines associated with T helper 2(Th 2). IL33 is a ligand for ST2(IL1RL1), and ST2 is an IL-1 family receptor highly expressed on Th2 cells, mast cells and group 2 innate lymphocytes. An exemplary complete cDNA sequence for human IL33 has accession number BC047085.1 and an exemplary amino acid sequence for human IL33 has accession number AAH 47085.1. The term "IL 33" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants.

The term "IL 18 BP" as used herein refers to interleukin-18 binding protein. The protein binds to IL18, prevents IL18 from binding to its receptor, and thus inhibits IL 18-induced IFN- γ production. The GENBANK accession number for an exemplary complete cDNA sequence of human IL18BP is BC044215.1 and the GENBANK accession number for an exemplary amino acid sequence of human IL18BP is AAH 44215.1. The term "IL 18 BP" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

In certain embodiments, the cytokine is a chemokine. In certain embodiments, the chemokine comprises a CC chemokine. In certain embodiments, the CC chemokine is selected from CCL4, CCL8, CCL6, CCL9, and CCL 11. In certain embodiments, the chemokine comprises a CXC chemokine. In certain embodiments, the CXC chemokine is selected from CXCL2, CXCL5, CXCL16, CXCL9 and CXCL 13.

Chemokines are a family of small cytokines or signaling proteins secreted by cells. Chemokines can be divided into four major subfamilies: CXC, CC, CX3C and XC, all of which exert their biological effects by interacting with G protein-linked transmembrane receptors (known as chemokine receptors) that are selectively present on the surface of target cells. Chemokines are responsible for the migration of basal leukocytes (homeostasis) or actively involved in the inflammatory response, attracting immune cells to sites of inflammation (inflammation).

As used herein, the term "CCL 4" refers to C-C motif chemokine 4, which is a single factor with inflammatory and chemokinetic properties. The GENBANK accession number for an exemplary complete cDNA sequence of human CCL4 is BC107433.1 and the GENBANK accession number for an exemplary amino acid sequence of human CCL4 is AAI 07434.1. The term "CCL 4" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "CCL 6" refers to chemokine (C-C motif) ligand 6, a small cytokine belonging to the CC chemokine family. The GENBANK accession number for an exemplary complete cDNA sequence of rat CCL6 is BC079460.1 and the GENBANK accession number for an exemplary amino acid sequence of rat CCL6 is AAH 79460.1. Human counterparts homologous to exemplary amino acid sequences can be found by BLAST, e.g., NCBI accession number: NP _665905.2 and NP _ 116741.2. The term "CCL 6" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "CCL 8" refers to chemokine (C-C motif) ligand 8, a small cytokine belonging to the CC chemokine family. It attracts monocytes, lymphocytes, basophils and eosinophils and may play a role in neoplasia and inflammatory host responses. The GENBANK accession number for an exemplary complete cDNA sequence of human CCL8 is BC126242.1 and the GENBANK accession number for an exemplary amino acid sequence of human CCL8 is AAI 26243.1. The term "CCL 8" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "CCL 9" refers to C-C motif chemokine 9, which is a single factor with inflammatory, pyrogenic, and chemokinetic properties. It circulates in the blood of healthy animals in high concentrations. It binds to high affinity receptors, activating calcium release in neutrophils. It also inhibits colony formation of bone marrow immature progenitor cells. An exemplary complete cDNA sequence of mouse CCL9 has a GI number of 85440457 and an exemplary amino acid sequence of mouse CCL9 has an NCBI accession number NP _ 035468.1. Human counterparts homologous to exemplary amino acid sequences can be found by BLAST, e.g., NCBI accession number: NP _665905.2 and NP _ 005055.3. The term "CCL 9" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

The term "CCL 11" as used herein refers to C-C motif chemokine 11, a small cytokine belonging to the CC chemokine family. CCL11 selectively recruits eosinophils by inducing chemotaxis and is therefore associated with allergic reactions. The GENBANK accession number for an exemplary complete cDNA sequence of human CCL11 is BC017850.1 and the GENBANK accession number for an exemplary amino acid sequence of human CCL11 is AAH 17850.1. The term "CCL 11" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "CXCL 2" refers to C-X-C motif chemokine 2, which is a hematopoietic chemokine that inhibits the proliferation of hematopoietic progenitor cells in vitro. The GENBANK accession number for an exemplary complete cDNA sequence of human CXCL2 is BC015753.1 and the GENBANK accession number for an exemplary amino acid sequence of human CXCL2 is AAH 15753.1. The term "CXCL 2" encompasses homologs in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "CXCL 5" refers to C-X-C motif chemokine 5, a small cytokine belonging to the CXC chemokine family. It is produced after the inflammatory cytokine interleukin-1 or tumor necrosis factor-alpha stimulates cells. The GENBANK accession number for an exemplary complete cDNA sequence of human CXCL5 is BC008376.1 and the GENBANK accession number for an exemplary amino acid sequence of human CXCL5 is AAH 08376.1. The term "CXCL 5" encompasses homologs in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "CXCL 9" refers to chemokine (C-X-C motif) ligand 9, a small cytokine belonging to the CXC chemokine family, also known as the interferon gamma (MIG) induced monokine. CXCL9 is a T cell chemokine induced by IFN- γ. The GENBANK accession number for an exemplary complete cDNA sequence of human CXCL9 is BC063122.1 and the GENBANK accession number for an exemplary amino acid sequence of human CXCL9 is AAH 63122.1. The term "CXCL 9" encompasses homologs in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

The term "CXCL 13" as used herein refers to chemokine (CXC motif) ligand 13, a small cytokine belonging to the CXC chemokine family, also known as B Lymphocyte Chemokine (BLC) or B cell attracting chemokine 1(BCA) -1, is a protein ligand encoded by the CXCL13 gene in humans. CXCL13 has selective chemotactic effects on B cells belonging to the B-1 and B-2 subsets and causes its effects by interacting with the chemokine receptor CXCR 5. CXCL13 and its receptor CXCR5 control the organization of B cells within lymphoid follicles and are highly expressed in the liver, spleen, lymph nodes and intestinal tract of humans. The CXCL13 gene is located on human chromosome 4 in other CXC chemokine clusters. In T cells, expression of CXCL13 is thought to reflect the germinal center origin of T cells, and in particular reflects a subset of T cells called T follicle helper cells (or TFH cells). The GENBANK accession number of an exemplary complete CDNA sequence for human CXCL13 is EF064743.1, and the GENBANK accession number of an exemplary amino acid sequence for human CXCL13 precursor is AAH 12589.1. The term "CXCL 13" encompasses homologs in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "CXCL 16" refers to chemokine (C-X-C motif) ligand 16, a small cytokine belonging to the CXC chemokine family. CXCL16 consists of a CXC chemokine domain, a mucin-like stem, a transmembrane domain and a cytoplasmic tail containing a potential tyrosine phosphorylation site that may bind SH 2. These are unusual features of chemokines and allow CXCL16 to be expressed with cell surface binding molecules and soluble chemokines. CXCL16 is typically produced by dendritic cells found in the T cell region of lymphoid organs as well as cells found in the red marrow of the spleen. Cells that bind and migrate in response to CXCL16 include several subsets of T cells, as well as natural killer T (nkt) cells. CXCL16 interacts with the chemokine receptor CXCR6 (also known as Bonzo). An exemplary mRNA sequence for human CXCL16 has accession number AF337812.1 and an exemplary amino acid sequence for human CXCL16 precursor has accession number AAK 38275.1. The term "CXCL 16" encompasses homologs in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

Cell Adhesion Molecules (CAMs) are a subset of cell adhesion proteins located on the cell surface that are involved in binding to other cells or the extracellular matrix (ECM) in processes related to cell adhesion. Cell adhesion molecules help cells adhere to each other and to the surrounding environment. CAMs include the cell adhesion molecules of the immunoglobulin superfamily (IgCAM), cadherins, integrins and the C-type lectin-like domain protein superfamily (CTLD). In certain embodiments, the cell adhesion molecule comprises ICAM1, CSF3r, Itgam, Siglecf, Adam8, Chl1, Sirpa, Nrcam, Emilin2, Emilin1, Tubb6, and Parvb.

As used herein, the term "ITGAM" refers to integrin α -M, which is involved in various adhesive interactions of monocytes, macrophages and granulocytes as well as mediating uptake of complement coated particles. The GENBANK accession number for an exemplary complete cDNA sequence of human ITGAM is BC096346.3 and the GENBANK accession number for an exemplary amino acid sequence of human ITGAM is AAH 96346.1. The term "ITGAM" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "sigleccf" refers to Ig-like lectin F that binds sialic acid, also known as Siglec5, which is a cell surface protein that binds sialic acid. It is mainly present on the surface of immune cells and is a subset of type I lectins. Exemplary complete cDNA sequence of mouse SIGLECF has accession number BC145038.1 and exemplary amino acid sequence of mouse SIGLECF has accession number AAI 45039.1. Human counterparts homologous to exemplary amino acid sequences can be found by BLAST, e.g., NCBI accession number: NP _003821.1 and XP _ 016882908.1. The term "sigleccf" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants.

As used herein, the term "ADAM 8" refers to a protein 8 containing integrin and metalloprotease domains that is a member of the ADAM (integrin and metalloprotease domain) family. Members of this family are membrane-fixed proteins, structurally related to snake venom double integrins, and have been implicated in a variety of biological processes involving cell-cell and cell-matrix interactions, including fertilization, muscle development and neurogenesis. The protein encoded by the gene may be involved in cell adhesion during neurodegenerative processes. An exemplary complete cDNA sequence of human ADAM8 has GENBANK accession No. D26579.1, and an exemplary amino acid sequence of human ADAM8 has GENBANK accession No. BAA 05626.1. The term "ADAM 8" encompasses homologs, variants obtained by proteolytic processing, splice variants, and allelic variants in other species.

As used herein, the term "CHL 1" refers to the neural cell adhesion molecule L1-like proteins, extracellular matrix and cell adhesion proteins, which play a role in the development of the nervous system and synaptic plasticity. Both soluble and membranous forms promote neurite outgrowth of cerebellar and hippocampal neurons and inhibit neuronal cell death. The GENBANK accession number for an exemplary complete cDNA sequence of human CHL1 is BC104918.1 and the GENBANK accession number for an exemplary amino acid sequence of human CHL1 is AAI 04919.1. The term "CHL 1" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "SIRPA" refers to an immunoglobulin-like cell surface receptor for signal-regulated protein alpha, also known as the tyrosine-protein phosphatase non-receptor type substrate 1, CD 47. It acts as a docking protein and induces the transfer of PTPN6, PTPN11 and other binding partners from the cytosol to the plasma membrane. Supporting adhesion of cerebellar neurons, neurite outgrowth and glial cell attachment. An exemplary complete cDNA sequence of human SIRPA has GENBANK accession number BC026692.1 and an exemplary amino acid sequence of human SIRPA has GENBANK accession number AAH 26692.1. The term "SIRPA" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants.

As used herein, the term "NRCAM" refers to a neuronal cell adhesion molecule, a homotypic glycoprotein expressed on the surface of neurons, glia, and skeletal muscle. It is essential for a normal response to contact of cells in the brain and peripheral nervous system. Cell Adhesion Molecules (CAMs) are members of the immunoglobulin superfamily. The GENBANK accession number for an exemplary complete cDNA sequence of human NRCAM is BC115736.1 and the GENBANK accession number for an exemplary amino acid sequence of human NRCAM is AAI 15737.1. The term "NRCAM" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants.

As used herein, the term "EMILIN 2" refers to elastin microfiber interface 2, which is a member of the EMILIN family of extracellular matrix glycoproteins. It has cell adhesion ability. The GENBANK accession number for an exemplary complete cDNA sequence of human EMILIN2 is BC136541.1, while the GENBANK accession number for an exemplary amino acid sequence of human EMILIN2 is AAI 36542.1. The term "EMILIN 2" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "EMILIN 1" refers to elastin microfiber interface 1, which is a member of the EMILIN family of extracellular matrix glycoproteins. It has cell adhesion ability. An exemplary complete cDNA sequence of human EMILIN1 is accession number BC136279.1 and an exemplary amino acid sequence of human EMILIN1 is accession number AAI 36280.1. The term "EMILIN 1" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants.

As used herein, the term "TUBB 6" refers to the tubulin β -6 chain, the major component of microtubules. It binds to two moles of GTP, one at exchangeable sites on the beta chain and the other at non-exchangeable sites on the alpha chain. The GENBANK accession number for an exemplary complete cDNA sequence of human TUBB6 is BC002654.1 and the GENBANK accession number for an exemplary amino acid sequence of human TUBB6 is AAH 02654.1. The term "TUBB 6" encompasses homologs of other species, variants obtained by proteolytic processing, splice variants, and allelic variants.

As used herein, the term "PARVB" refers to β -parvin, an adaptor protein that plays a role in integrin signaling via ILK and in activating GTPases CDC42 and RAC1 by guanine exchange factors. It is involved in the reorganization of the actin cytoskeleton and the formation of sheet lipoproteins. It plays a role in cell adhesion, cell spreading, establishment or maintenance of cell polarity, and cell migration. An exemplary complete cDNA sequence of mouse PARVB has accession number AF237770.1, while an exemplary accession number of amino acid sequence of mouse PARVB is AAG 27172.1. Human counterparts homologous to exemplary amino acid sequences can be found by BLAST, e.g., NCBI accession number: NP _001003828.1 and NP _ 037459.2. The term "PARVB" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "ICAM 1" refers to intercellular adhesion molecule 1, a cell surface glycoprotein, commonly expressed on endothelial cells and cells of the immune system. It binds to integrins of the CD11a/CD18 or CD11b/CD18 type and is utilized by rhinoviruses as receptors for entry into the respiratory epithelium. An exemplary complete cDNA sequence of human ICAM1 has accession number BC015969.2 and an exemplary amino acid sequence of human ICAM1 has accession number AAH 15969.1. The term "ICAM 1" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "CSF 3R" refers to colony stimulating factor 3 receptor, which is a receptor for granulocyte colony stimulating factor (CSF3) necessary for granulocyte maturation. It plays a crucial role in the proliferation, differentiation and survival of the neutrophil line along the cell. In addition, it may play a role in certain adhesion or recognition events on the cell surface. The GENBANK accession number for an exemplary complete cDNA sequence of human CSF3R is BC053585.1 and the GENBANK accession number for an exemplary amino acid sequence of human CSF3R is AAH 53585.1. The term "CSF 3R" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants thereof.

As used herein, the term "IL 2" refers to interleukin 2, a cytokine signaling molecule in the immune system. It is a protein that regulates the activity of the white blood cells (leukocytes, usually lymphocytes, such as B cells) responsible for immunity. IL2 mediates its effects by binding to IL2 receptors expressed by lymphocytes. IL2 has been reported to induce T cells (Lan et al, Journal of Autoimmunity,2008,31(1):7-12), B cells (Karray et al, J Exp Med.1988, 7.1/month; 168(1):85-94) and dendritic cells. The GENBANK accession number for an exemplary complete cDNA sequence of human IL2 is AH002842.2 and the GENBANK accession number for an exemplary amino acid sequence of human IL2 is AAD 48509.1. The term "IL 2" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants.

As used herein, the term "IL 21" refers to interleukin-21, which is also a cytokine that co-stimulates T and Natural Killer (NK) cell proliferation and function and regulates B cell survival and differentiation as well as dendritic cell function (see Croce et al, J Immunol Res.2015; 2015: 696578). The GENBANK accession number for an exemplary complete cDNA sequence of human IL21 is NM _021803.3 and the GENBANK accession number for an exemplary amino acid sequence of human IL21 is NP _ 068575.1. The term "IL 21" encompasses homologues in other species, variants obtained by proteolytic processing, splice variants and allelic variants.

The "inducible T cell costimulators (ICOS)" are also known as "AILIM", "CD 278" and "MGC 39850". The GENBANK accession number for an exemplary complete cDNA sequence of ICOS is NM _012092.3, while the GENBANK accession number for an exemplary amino acid sequence of human ICOS is NP _ 036224. ICOS belongs to the family of CD28 and CTLA-4 cell surface receptors, and is homologous to CD28 and CTLA-4. It forms homodimers through disulfide bonds and plays an important role in cell signaling, immune response and regulation of cell proliferation through PI3K and AKT pathways during germinal center formation, T/B cell cooperation and immunoglobulin class switching. ICOS, together with CD28 and CTLA-4, is expressed on activated CD4 and CD 8T cells, and has a potential role in modulating adaptive T cell responses (e.g., activation and proliferation of T cells). Unlike CD28, which is constitutively expressed on T cells and provides the costimulatory signals required to fully activate resting T cells, ICOS is only expressed following initial T cell activation. ICOS also plays a role in the development and function of other T cell subsets, including Th1, Th2, and Th 17. ICOS co-stimulates T cell proliferation and cytokine secretion associated with Th1 and Th2 cells. Among the various disease models, ICOS Knockout (KO) mice have impaired development of autoimmune phenotypes, including diabetes (Th1), airway inflammation (Th2), and EAE neuroinflammation model (Th 17). In addition to its role in regulating T effector (Teff) cell function, ICOS also regulates T regulatory cells (Tregs). Furthermore, ICOS is expressed at high levels on tregs and is involved in Treg homeostasis and function (see US patent application US 20160304610). The role of ICOS in promoting CD4+ T cell proliferation was suggested to be independent of IL-2 signaling (see Wikenheiser DJ and Stumhofer JS, ICOS co-stimulation: friend or foe.

Agonists of ICOS (e.g., ICOSL) bind to the extracellular domain of ICOS, activating ICOS signaling, thereby increasing T cell activation and proliferation.

As used herein, the term "ICOS ligand (ICOSL)" also refers to "B7H 2", "GL 50", "B7-H2", "B7 RP 1", "CD 275", "ICOSLG", "LICOS", "B7 RP" costimulatory molecule-1 of the B7 superfamily, "ICOS-L" and "KIAA 0653" play a positive role in the immune response. The GENBANK accession number for an exemplary complete cDNA sequence of ICOSL is NM _015259.5, while the GENBANK accession number for an exemplary amino acid sequence of human ICOSL is NP _ 056074.1. ICOSL shares 19-20% sequence identity with CD80/CD86 and is secreted or expressed as a cell surface protein. Human ICOSL has two splice variants (hGL50 and B7-H2/B7RP-1/hLICOS), both of which have the same extracellular domain but differ at the carboxy terminus of their cytoplasmic regions. In humans, ICOSL is expressed on B cells, dendritic cells, monocytes/macrophages and T cells. Unlike CD80/CD86, ICOSL does not interact with CD28 or CTLA-4(CD152), but acts as a non-covalently linked homodimer on the cell surface and binds ICOS. Human ICOSL has been reported to bind to human CD28 and CTLA-4 (see US patent application US 20160304610).

The ICOS/ICOS-L interaction is associated with a T cell mediated immune response in vivo. In addition, defects in ICOS lead to impaired Germinal Center (GC) formation (reduced GC number and size), isotype class switch defects in T-cell dependent B-cell responses, and IL-4 and IL-13 defects. Production (see Khayyamian et al, ICOS ligand expressed on human endothelial cells costimulates Th1 and Th2 cytokine secretion by memory CD 4T cells, PNAS, Vol.9, No.9,2002, 6198-. In GC, long-lived plasma cells (LLPC) and Memory B Cells (MBC) undergo class switching and somatic hypermutation to increase antibody affinity.

In certain embodiments, culturing PBMCs in the presence of ICOS or ICOSL may increase the total amount of antibody or antigen-binding fragment thereof produced by the PBMCs.

Agonists of ICOS can be screened by determining their affinity and binding specificity. Methods for determining the affinity and specificity of binding, such as competitive and non-competitive binding assays, are known in the art, including ELISA, RIA, flow cytometry, and the like. ICOS agonist effects can be measured by the activation of T cells by ICOS. T cell activation can be measured by detecting CD4+ T cell proliferation, cell cycle progression, cytokine (e.g., IL-2) release, CD25 and CD69 upregulation, and the like.

ICOS agonists include compounds or proteins such as the agonist antibodies JTX-2011 (joint Therapeutics Inc) and GSK3359609(GSK), as well as antibodies described in U.S. patent applications US20160304610, US 20170174767, and WO 2012/131004.

CD40L, also referred to herein as CD40 ligand or CD154, is a protein that is expressed predominantly on activated T cells (hereafter its expression has been found in a variety of cells, including platelets, mast cells, macrophages, basophils, NK cells, B lymphocytes, and non-hematopoietic cells), and is a member of the TNF superfamily of molecules. It binds to CD40 on Antigen Presenting Cells (APCs) and acts as a costimulatory molecule, which is particularly important on a subset of T cells called T follicle helper cells (TFH cells). On TFH cells, CD40L promotes B cell maturation and function by binding CD40 to the B cell surface, thereby facilitating communication between cells. The GENBANK accession number for the exemplary complete cDNA sequence of CD40L is NM _000074.2, while the GENBANK accession number for the exemplary amino acid sequence of human CD40L is NP _ 000065.1.

As used herein, the phrase "B cell activating factor" or "BAFF" refers to a tumor necrosis family ligand, such as a TNF family ligand. BAFF is expressed on the cell surface and, as a regulatory protein, participates in the interaction between membrane surface proteins on immune cells such as B cells. Secretion of BAFF is a potent B cell growth factor that can help B cells proliferate and act as a costimulator. BAFF has been reported to be critical for the survival of cells secreting antibodies from memory cells (Avery DV et al, J Clin Invest,2003,112: 286-97). An exemplary amino acid sequence of human CD40L has GENBANK accession number AAD 25356.1.

"OX 40L" is a ligand of OX40(CD134) and is expressed on cells such as DC2s (a subset of dendritic cells), enabling expansion of Th2 cell differentiation. OX40L is also known as CD252 (cluster of differentiation 252). It has been reported that OX40 is co-expressed with ICOS in T follicular helper cells (Tfh) and affects the interaction between Tfh cells and B cells in Germinal Centers (GC), thereby affecting the development of B cells and the maturation of plasma cells in plasma cells. The GENBANK accession number of an exemplary cDNA sequence of human OX40L is AJ277151.1, and the GENBANK accession number of an exemplary amino acid sequence of human OX40L is CAB 9654.3.

The term "Toll-like receptors (TLRs)" is a family of proteins that play a key role in the innate immune system (non-specific immunity). They are single, transmembrane non-catalytic receptors, usually expressed on sentinel cells (e.g., macrophages and dendritic cells), that recognize structurally conserved molecules derived from microorganisms. In addition to the extracellular and transmembrane domains, TLRs contain a cytoplasmic Toll-interleukin 1 receptor resistance (TIR) domain. Once these microorganisms break through physical barriers such as skin or intestinal mucosa, they are recognized by TLRs, which activate immune cell responses. TLRs recognize highly conserved structural motifs, pathogen-associated molecular patterns (PAMPs), which are expressed only by microbial pathogens, such as Lipopolysaccharide (LPS) of gram-negative bacteria and lipoprotein acids (LTA) of gram-positive bacteria and flagellin, among others, or risk-associated molecular patterns (DAMPs), which are endogenous molecules released from necrotic or dying cells. Many tumor cells undergo immune system mediated necrosis and may lead to further activation of the inflammatory response through TLRs. The human TLR family includes TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10 expressed on various immune cell types. The mouse TLR series includes TLR1-9 and TLR 11-13.

As used herein, "Toll-like receptor ligand" refers to an agonist or antagonist of a Toll-like receptor. In certain embodiments, the TLR ligand is an agonist, such as a pathogen-associated molecular pattern (PAMP). Examples of TLR agonists that activate TLRs include, but are not limited to, imiquimod, GS-9620(Gilead, see Roethle et al, 2013), compound 32(GSK2245035, GSK, see Biggadike et al, 2016) and risimod (R848), imidazoquinoline, nucleic acids comprising CpG ODNs (e.g., unmethylated CpG dinucleotides (e.g., ODN2216) and poly I: C, monophosphoryl lipid a (mpla) or other lipopolysaccharide derivatives, single or double stranded RNA, flagellin, poly-dipeptides), TSLP, Tumor Necrosis Factor (TNF) α, type I interferons (e.g., IFN α/β), type II interferons (e.g., IFNy), lipids, avastin, EFNB1, EPHB4, protein B2, axon-targeting protein 4C, BLIMP-1, and IRF 4. (see J.Med.chem.Roethle et al, 2013. identification and optimization of Poterin ketone Toll-like receptor 7(TLR7) agonists for oral treatment of viral hepatitis, J.Med.chem.Biggadike et al, 2016.59,1711-1726. discovery of a highly potent and selective intranasal Toll-like receptor 7 agonist 6-amino-2- { [ (1S) -1-methylbutyl ] oxy } -9- [5- (1-piperidinyl) pentyl ] -7, 9-dihydro-8H-purin-8-one (GSK 2245) for treatment of asthma)

TLR agonists specific for TLR types are reported in the literature, for example BCG (TLRs 1, 2,4 and 6), lipopeptides (TLRs 1, 2 and 6), monophosphoryl lipid a (mpl), LPS, RC529, AS01, AS02, AS04 and glucopyranosyl lipid adjuvant (GLA-SE) (TLR4), poly (I: C) (TLR3), flagellin (TLR5), single-and R484/rasimod (TLR7 and TLR8) or double-stranded (ds) RNA (TLR3), imiquimod and type 1 interferon (TLR7), and DNA containing CpG motifs, AS15 and IC31(TLR 9). Endogenous molecules released from stressed or dead cells, such as heat shock proteins (HSP, TLR2 and TLR4) and high mobility group 1 boxes (HMGB1, TLR2 and TLR4), are also considered important TLR agonists (see Deng Sl et al, progress in the study of the role of toll-like receptors and TLR agonists in human brain glioma immunotherapy, (see Protein Cell 2014,5(12): 899-911, Zhang WW AND Matlashewski G, Toll-like receptor 7 AND/or 8 agonist vaccine adjuvant immunization enhances the protective IMMUNITY of BALB/c mice against Leishmania, INFECTION AND IMMUNITY, Aug.2008, p.3777-3783, Gauwelaert ND et al, TLR4 agonist vaccine adjuvant GLA-SE requires a typical AND atypical TH1 induction mechanism of action, PLoS one. Jan5, 11(1): e0146372, Maizoneuve C et al, potential to release NOD and Toll-like agonists as vaccine adjuvants Proc Natl Acad Sci U S A.2014Aug 26; 111(34): 12294-9).

In certain embodiments, wherein the antibody-enhancing composition is added to the culture medium at the beginning of the culture, or after 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days. In certain embodiments, wherein the antibody-enhancing composition removes the composition from the culture medium after 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days.

In certain embodiments, wherein the antibody-potentiating composition exhibits a synergistic effect in stimulating antibody production in vitro with two or more antibody potentiating factors of the antibody-potentiating composition. The two or more antibody enhancing factors may be selected from ADSP, CD40L, ICOSL, ICOS, TLR agonists, co-stimulators (CD40, CD40L, ICOSL, ICOS, APRIL, TNF family B cell activating factor (BAFF), OX40 or OX40L), toll-like receptor (TLR) agonists (TLR1 agonists, TLR2 agonists, TLR3 agonists, TLR4 agonists, TLR5 agonists, TLR6 agonists, TLR7 agonists, TLR8 agonist and TLR9 agonist), CpG oligo/deoxyribonucleic acid or K CpG), anti-apoptotic proteins (Bcl-2, Bcl-6, Bcl-XL, Bcl-w, Mcl-1 or analogs thereof), TNF, type I interferons (e.g., IFN-. alpha IFN-. beta., IFN-. epsilon., IFN-. kappa.and IFN-. omega.), type II interferons (e.g., IFN. gamma.), lipids, avasimid, EFNB1, EPHB4, protein B2, axon-targeting protein 4C, BLIMP-1 and IRF 4.

In vitro antibody production

The present disclosure provides a method of producing an antibody or antigen-binding fragment thereof that specifically binds to an antigen of interest, the method comprising mixing the antigen, an antibody-producing cell composition (AGC) and an antibody enhancing composition in a culture medium to form a mixture, culturing the mixture

(ii) incubating the mixture in the presence of a nutrient,

wherein the AGC comprises at least one B cell and at least one other type of cell derived from Peripheral Blood Mononuclear Cells (PBMCs), and the antibody enhancing composition comprises one or more adipose tissue-derived secreted proteins (ADSPs). In certain embodiments, the antibody enhancing composition further comprises IL2 and/or IL 21.

In certain embodiments, wherein said B cells prior to culture are immature. In certain embodiments, wherein the B cells prior to said culturing do not produce antibodies to the antigen of interest.

The present disclosure also provides a method of producing an antibody or antigen-binding fragment thereof that specifically binds to an antigen of interest, the method comprising:

culturing the mixture

wherein the AGC comprises at least one B cell and at least one other type of cell derived from Peripheral Blood Mononuclear Cells (PBMCs), and the composition of enhanced antibodies comprises IL2, IL21, and one or more adipose tissue-derived secreted proteins (ADSPs)).

The ADSP, IL2 and IL21 may be introduced into the culture medium separately, either at once or in any suitable order. IL-2 and/or IL21 can promote proliferation of B cells, and ADSP and antigen can stimulate B cells to produce antibodies, and IL21 can also promote class switching of antibodies from IgM to IgG. In certain embodiments, IL2 is added to the culture medium first, followed by the antigen and ADSP, followed by IL 21.

In certain embodiments, the addition of ADSP to the culture medium produces antibodies with antigen specificity. In certain embodiments, the ADSPs include cytokines, such as IL1 β, IL1f9, IL10, IL27, IL33, IL18BP, chemokines, such as CCL8, CCL4, CXCL2, CCL6, CCL9, CCL11, CXCL5, CXCL2, and CXCL9, and cell adhesion molecules, such as ICAM1, CSF3r, Itgam, Siglecf, Adam8, Chl, Sirpa, Nrcam, Emilin2, Emilin1, Tubb6, and Parvb. In certain embodiments, the ADSPs include IL1 β, CCL8, and CXCL 5. In certain embodiments, an antibody or antigen-binding fragment produced according to the methods provided herein specifically binds human and/or non-human antigens with a binding affinity (KD) of about 0.01nM to about 100nM, about 0.1nM to about 100nM, about 0.01nM to about 10nM, about 0.1nM to about 10nM, about 0.01nM to about 5nM, about 0.1nM to about 5nM, about 0.01nM to about 1nM, about 0.1nM to about 1nM, or about 0.01nM to about 0.1 nM.

The first group of ADSPs may be added to the culture medium during a first period after the start of the culturing, and then the second group of ADSPs may be added to the culture medium during a second period. In certain embodiments, the first set of ADSPs is removed prior to adding the second set of ADSPs. In certain embodiments, the second set of ADSPs is removed from the mixture at the end of the second time period. In certain embodiments, the "first period" or "second period" refers to, for example, 0 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, a month, or more. In some embodiments, the "first period" or the "second period" have the same or different lengths (or spans) of time. In certain embodiments, the ADSPs of the first and/or second groups are present in the mixture for 0 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days after the start of cultivation for 4 days. In certain embodiments, the first and second sets of ADSPs are the same or different.

In certain embodiments, wherein the first or second set of ADSPs comprises cytokines, such as IL1 β, IL1f9, IL10, IL27, IL33, IL18BP chemokines, such as CCL8, CCL4, CXCL2, CCL6, CCL9, CCL11, CXCL5, CXCL2, and CXCL9, and cell adhesion molecules, such as ICAM1, CSF3r, Itgam, Siglecf, Adam8, Chl, Sirpa, Nrcam, Emilin2, Emilin1, Tubb6, and Parvb.

The ADSP is present in the mixture at a concentration of at least 0.5, 1, 2,3, 4,5, 6, 7,8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 1500, 2000, 3000, 4000, 5000 or more ng/ml, or 0.5, 1, 2,3, 4,5, 6, 7,8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 1500, 2000, 3000, 4000, 5000 or more ml, or 0.1, 0.2, 0.5, 1, 2,3, 4,5, 8, 30, 5000, 25, 30, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 1000, 1500, 2000, 3000, 4000, 5000 or more ml, or 0.1, 0.2, 0.5, 1, 2,3, 4,6, 5, 6, 5, 8, 30, 5, 30, 5, 30, 5, 6, 5, 6, 5, 9, 5, 30, 5, 6, 5, 6,9, 5, 40, 5, 9, 5, 30, 5, 9, 5, 30, 5, 9, 5, or more ml, 6, or more ml of an, 65. 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, or more nM.

In certain embodiments, IL1 β is present in the mixture at a concentration of 40, 400, or 1000 ng/ml. In certain embodiments, CCL8 is present in the mixture at a concentration of 10, 50, or 100 ng/ml. In certain embodiments, CXCL5 is present in the mixture at a concentration of 1, 10, or 20 ng/ml. In certain embodiments, CCL4 is present in the mixture at a concentration of 5 ng/ml. In certain embodiments, CXCL2 is present in the mixture at a concentration of 10 ng/ml. In certain embodiments, CXCL16 is present in the mixture at a concentration of 2 ng/ml.

In certain embodiments, IL2 is present in the mixture for 0 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, one month, or more. In certain embodiments, IL21 is present in the mixture for 0 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, one month, or more. In certain embodiments, 0 hour, 0.5 hour, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days after the start of cultivation, IL2 and/or IL21 are present in the mixture.

In certain embodiments, IL2 is present in an amount of at least 0.5, 1, 2,3, 4,5, 6, 7,8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, or higher ng/ml, or in an amount of 0.5, 1, 2,3, 4,5, 6, 7,8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, or higher μ g/ml, or in an amount of 0.1, 0.2, 3, 4,5, 6, 7,8, 9, 10, 12, 15, 20, 25, 50, 1, 2,3, 4,5, 6, 7,8, 9, 10, 12, 25, 50, 60,65, 50, 85, 90, or higher μ g/ml, or higher, 250. 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000 or more nM are present in the mixture.

In certain embodiments, IL21 is present in an amount of at least 0.5, 1, 2,3, 4,5, 6, 7,8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 1000, or higher ng/ml, or 0.5, 1, 2,3, 4,5, 6, 7,8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60,65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, or higher μ g/ml, or 0.1, 0.2, 0.5, 1, 2,3, 4,5, 6, 7,8, 30, 65, 70, 50, 80, 90, 50, 40, 5, 6, 5, 10, 30, 50, 5, 50, 70, 50, 5, or more μ g/ml 95. 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000 or more nM.

In certain embodiments, IL2 and IL21 are present in a ratio of 1: 1. 1: 2. 1: 5. 1: 10. 1: 20. 1: 30. 1: 40. 1: 50. 1: 60. 1: 70. 1: 80. 1: 90. 1: 100. 1: 150. 1: 200. 1: 500. 1: 1000. 1: 2000. 1: 5000. 1: 10000 or 1: 20000 are present in the mixture.

In certain embodiments, wherein IL2 is present in the mixture at a concentration of 10 ng/ml. In certain embodiments, wherein IL21 is present in the mixture at a concentration of 50 ng/ml.

In certain embodiments, wherein the mixture further comprises an antigen. The antigen is present in the mixture at the beginning of the culture, or 1, 2,3, 4,5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 days after the start of planting. In certain embodiments, wherein the antigen is present in the mixture for at least 0.5 day, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, a month, or more.

The present disclosure provides a method of inducing proliferation of an antibody-producing cell composition (AGC), B cell activation and differentiation, B cell maturation, and/or promoting class switching in AGC to produce IgG, wherein the method comprises culturing the AGC in a medium comprising IL2, ADSP, and/or IL 21. In certain embodiments, the AGC comprises PBMCs.

AGC or PBMCs can be cultured in vitro according to protocols known in the art (Panda, s.k. and Ravindran, B. (2013). in vitro culture of human PBMCs Bio-protocol 3(3): e 322.). After stimulation, the proliferation of AGC or PBMC can be measured using methods commonly used in the art, such as flow cytometry.

B cell activation and differentiation is the process by which peripheral blood B lymphocytes undergo antigen-induced activation and differentiation. Activated B cells can increase the proportion of antibody-secreting plasma cells or memory B cells. Class switching occurs at the plasma cell stage. B cells may first differentiate into plasmablast-like cells and then into plasma cells, which are then produced during infection, and their antibodies have higher affinity (GC) and produce more antibodies than plasmablasts due to affinity maturation of germinal centers as compared to plasmablasts (see Nutt et al, Nature Reviews immunology.2015,15(3): 160). Plasma cells are usually caused by germinal center reactions caused by T cell-dependent (TD) B cell activation, but they may also be caused by T cell-dependent (TI) B cell activation (see Bortnick et al, The Journal of immunology.188(11): 5389-5396)). B cell activation or differentiation can be detected or confirmed in vitro by methods known in the art, for example, by cell labeling with CD19, IgM, IgD, IgA antibodies and cell sorting using FACS. Memory B cells can be identified as CD19+ IgM-IgA-IgD-, while IgG-producing B cells are identified as CD19+ IgG +.

B-cell development is the differentiation of lymphoid precursor cells into the earliest unique B lineage cells (progenitor B cells (pro-B cells)) that express the transmembrane tyrosine phosphatase CD45R (or B220 in mice). proliferation and differentiation of pro-B cells into precursor B cells (pre-B cells) requires a microenvironment provided by bone marrow stromal cells that interact directly with pro-B and pre-B cells and secrete various cytokines, particularly IL-7, that support the developmental process.

The maturation phase of B cells depends on the rearrangement of immunoglobulin DNA in lymphoid stem cells. During B cell development, successive Ig-give rearrangements convert pro-B cells into immature B cells expressing mIgM with a single antigen specificity. Mature naive B cells will then be formed that still have a single specificity, while expressing mIgM and mIgD. Only pre-B cells capable of expressing membrane-bound μ heavy and surrogate light chains can progress along the developmental pathway. After generation of potent pre-B cell receptors, each pre-B cell undergoes multiple cell divisions, perhaps 6 to 8, yielding up to 256 generations. Each of these progeny pre-B cells can then rearrange different light chain gene segments, thereby increasing the overall diversity of the antibody library. In certain embodiments, B cell maturation occurs in the periphery. B cell maturation can be detected or confirmed in vitro by methods known in the art, for example, by detecting B cell surface markers, e.g., immature B cells express mIgM and migdd, while mature B cells express mIgG, mIgA and migdd. One skilled in the art will appreciate that methods such as cell staining and cell sorting with labeled antibodies to the above markers can be used.

Class switching is also known as isotype switching, isotype switching or Class Switching Recombination (CSR). This is a biological mechanism that changes the immunoglobulins (antibodies) produced by B cells from one type to another, for example from isotype IgM to isotypes IgG and IgE. In this process, the constant region portion of the antibody heavy chain is changed, but the variable region of the heavy chain remains unchanged. Since the variable region is unchanged, class switching does not affect antigen specificity. In contrast, antibodies retain affinity for the same antigen, but can interact with different effector molecules (see Honjo et al, Immunity,01Jun 2004,20(6): 659-668). Methods for determining IgG and IgM and levels thereof are known in the art, e.g., by ELISA using antibodies specific for the isotype.

In certain embodiments, an antibody produced according to the methods provided herein comprises IgG. In certain embodiments, an antibody involved according to the methods provided herein comprises an increase in the percentage of IgG.

In certain embodiments, wherein the method further comprises isolating the antibody from the mixture. For example, wherein the method further comprises fusing the antibody-producing B cell with an immortal cell line to obtain a hybridoma. In certain embodiments, wherein the method further comprises isolating RNA of the antibody gene and cloning a library of single domain antibodies using molecular biology techniques known in the art, followed by selection using phage display.

The term "hybridoma" as used herein refers to a fused hybrid cell in the course of hybridoma technology, wherein the method is used to produce large quantities of monoclonal antibodies. B cells that produce antibodies in response to an immune response are collected and then fused with immortal B cell cancer cells, myeloma, to produce hybrid cell lines known as hybridomas that have the antibody-producing ability of B-, the longevity and reproductive ability of myeloma. Hybridomas can be grown in cultures, each starting from one live hybridoma cell, and the resulting cultures are composed of genetically identical hybridomas that produce one antibody per culture (monoclonal) rather than a mixture of different antibodies (polyclonal). The monoclonal antibodies produced by each hybridoma cell line are chemically identical, as compared to polyclonal antibodies which are a mixture of many different antibody molecules.

The technique of selecting "phage display libraries" refers to a method of cloning VH and VL gene libraries separately by Polymerase Chain Reaction (PCR) and randomly recombining in a phage library, and then screening for antigen-binding phage, as described in Winter, G.et al, Ann.Rev.Immunol.12(1994) 433-455. Phage typically display antibody fragments as single chain fv (scFv) fragments or Fab fragments. Libraries from immune sources (e.g., antibody-producing PBMCs prepared by the methods provided herein) provide antibodies with high affinity for antigens without the need to construct hybridomas. Similarly, cloning of natural libraries (e.g., from humans) to obtain a single source of antibodies to a wide range of non-self as well as self antigens, as described by Griffiths, A.D., et al, EMBO J.12(1993) 725-. Finally, natural libraries can also be synthesized by cloning unrearranged V gene fragments in stem cells and using PCR primers comprising random sequences to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as suggested by Hoogenboom, H.R., and Winter, G., J.mol.biol.227(1992)381-388, and the like. Patent publications describing human antibody phage libraries include, for example: U.S. Pat. nos. US 5,750,373, US 2005/0079574, US 2005/0119455, US 2005/0266000, US 2007/0117126, US 2007/0160598, US 2007/0237764, US 2007/0292936 and US 2009/0002360. Similar display libraries include ribosome display, yeast display, bacterial display, baculovirus display, mammalian cell display or mRNA display libraries (see, e.g., U.S. Pat. No.7,244,592; Chao et al, Nature protocols.1: 755-. These display methods are all conventional in the art, the specific manipulations of which can be found in the corresponding textbooks or operating manuals, see for example Mondon P et al, front.biosci.13: 1117-.

In certain embodiments, wherein the method further comprises obtaining a nucleic acid molecule that encodes a variable region of an antibody within the mixture. In certain embodiments, wherein the method further comprises introducing the nucleic acid molecule into a host cell under conditions suitable for expression of the antibody or antigen-binding fragment thereof. In certain embodiments, wherein the host cell is a CHO cell.

As used herein, the term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in either single-or double-stranded form, and polymers thereof. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. In particular, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-.

The nucleic acid sequences encoding the variable regions of the antibodies or antigen-binding fragments thereof are transformed into host cells to produce the antibodies and cultured in appropriately optimized conventional nutrient media to induce promoters, select transformants or amplify the sequences encoding the desired sequences. In another embodiment, the antibody or antigen-binding fragment thereof can be produced by homologous recombination as known in the art.

In certain embodiments, wherein the method further comprises obtaining a nucleic acid sequence encoding a variable region of an antibody produced from the mixture.

DNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). The coding DNA may also be obtained synthetically.

In certain embodiments, wherein the method further comprises assessing whether the antibody or antigen-binding fragment thereof so produced specifically binds to an antigen of interest.

In certain embodiments, the assessment of binding specificity is performed by an immunoassay, such as an ELISA or a fluorescent immunoassay.

In certain embodiments, the assessment of binding specificity is determined by binding affinity. The binding affinity of an antibody or antigen-binding fragment thereof produced according to the methods provided herein can be represented by the KD value. KD as used herein refers to the ratio of off-rate to associated rate (koff/kon) and can be determined by using any conventional method known in the art, including but not limited to surface plasmon laser resonance methods, microscale thermophoresis, HPLC-MS methods, and flow cytometry (e.g., FACS) methods. In certain embodiments, the KD value can be suitably determined by using flow cytometry methods. A variety of immunoassay formats can be used to select antibodies specifically immunoreactive with a particular protein. For example, solid phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow & Lane, antibodies used, laboratory manual (1998), description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Typically, the specific or selective binding reaction produces a signal that is at least twice the background signal, more typically at least 10 to 100 times the background signal.

Alternatively, the binding affinity of antibodies and antigen-binding fragments thereof produced according to the methods provided herein for certain antigens can also be expressed in terms of "half maximal effective concentration" (EC50), which refers to the concentration of the antibody. 50% of its maximal effect (e.g. binding) was observed. EC50 values can be measured by methods known in the art, for example, sandwich assays such as ELISA, western blots, flow cytometry assays, and other binding assays.

Compositions for in vitro immunization

The disclosure herein provides compositions comprising an antibody-producing cell composition (AGC) comprising at least one B cell and at least one other type of cell vehicle derived from Peripheral Blood Mononuclear Cells (PBMCs).

In certain embodiments, the composition further comprises an antigen of interest. In certain embodiments, the antibody enhancing composition further comprises IL2 and/or IL 21.

For example, in certain embodiments, the AGC comprises at least one B cell and at least one T cell (e.g., T follicular helper cell). In certain embodiments, the AGC comprises at least one B cell and at least one dendritic cell. In certain embodiments, the AGC comprises at least one B cell, at least one T cell (e.g., T follicular helper cell), and at least one dendritic cell. In certain embodiments, the AGC comprises at least one B cell and at least one NK cell. In certain embodiments, the AGC comprises at least one B cell and at least one monocyte. In certain embodiments, the AGC comprises at least one B cell, T cell (e.g., T follicular helper cell) and at least one NK cell. In certain embodiments, the AGC comprises at least one B cell, at least one T cell (e.g., T follicular helper cell), at least one dendritic cell, and at least one NK cell.

In certain embodiments, the AGC further comprises adipocytes. In certain embodiments, the AGC comprises at least one adipocyte and B cell. In certain embodiments, the AGC comprises at least one adipocyte, at least one B cell, and at least one T cell (e.g., T follicular helper cell). In certain embodiments, the AGC comprises at least one adipocyte, at least one B cell, and at least one dendritic cell. In certain embodiments, the AGC comprises at least one adipocyte, B cell, T cell (e.g., T follicular helper cell), and at least one dendritic cell.

In certain embodiments, at least one of the B cell, T follicular helper cell, dendritic cell and adipocyte cell is a human cell. In certain embodiments, the B cell is a human B cell. In certain embodiments, the PBMCs are derived from human PBMCs.

Isolated antibody enhancers include the antibody enhancers previously described in this disclosure. In certain embodiments, the isolated antibody enhancing factor comprises the isolated ADSP, CD40L, ICOSL, ICOS, TLR agonists previously described.

The culture medium contained in the composition provides conditions suitable for the antibody-producing cell composition (AGC) to express an antibody or antigen-binding fragment thereof against the antigen of interest. The culture medium may be solid, liquid or semi-solid, intended to support the micro-vegetative bacteria or to provide essential nutrients (amino acids, carbohydrates, vitamins, minerals), growth factors, hormones and gases (CO2, O2) to maintain the growth of the cells and to regulate the physicochemical environment of the cells (pH buffer, osmotic pressure, temperature). Commercially available media such as Ham's F10(Sigma), Minimal Essential Medium (MEM) (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium (DMEM) (Sigma) are suitable for culturing the antibody-producing cell composition (AGC). Also meth.Enz.58:44(1979), Barnes et al, anal. biochem.102:255(1980), U.S. Pat. No.4,767, 704; 4,657,866, respectively; 4,927,762, respectively; 4,560,655, respectively; or 5,122,469; WO 90/03430; WO 87/00195; or one of the media described in U.S. Pat. No. re.30,985 et al, can be used as the medium for the antibody-producing cell composition (AGC). Any of these media may be supplemented as needed with hormones and/or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCINTM drugs), trace elements (defined as inorganic compounds typically present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements at appropriate concentrations known to those skilled in the art are also included. Culture conditions, such as temperature, pH, etc., are those previously used with the antibody-producing cell composition (AGC) selected for expression, and will be apparent to the ordinarily skilled artisan.

Identification of antibody enhancers for in vitro immunization

The disclosure herein provides a method of identifying an antibody enhancer for in vitro immunization, the method comprising:

a) isolating total RNA from cells derived from lymph nodes of an animal immunized with an antigen of interest;

c) culturing PBMCs in a culture medium comprising the antigen of interest, IL2, IL21 and a protein;

In certain embodiments, the cell is an adipocyte, T follicular helper cell, B cell, or dendritic cell. In certain embodiments, wherein the protein is expressed by adipocytes, T follicular helper cells, B cells, or dendritic cells. In certain embodiments, wherein the protein enhances IgG production.

Chimeric Antigen Receptor (CAR)

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid protein or polypeptide comprising an antigen binding domain (e.g., a single chain variable fragment (scFv)) of a T-linked antibody. Cell signaling or T cell activation domains (see, e.g., Kershaw et al, supra, Eshhar et al, Proc. Natl. Acad. Sci. USA,90(2): 720-. Using the antigen binding properties of monoclonal antibodies, CARs are able to redirect T cell specificity and reactivity to selected targets in a non-MHC-restricted manner. non-MHC restricted antigen recognition confers the ability of CAR-expressing T cells to recognize antigen independently of antigen processing, thereby bypassing the major mechanism of tumor escape. In addition, when the CAR is expressed in T cells, it does not dimerize with endogenous T Cell Receptor (TCR) alpha and beta chains

In certain embodiments, the CAR sequence comprises an antigen binding domain, e.g., a VH and VL gene segment of an antibody made according to the methods provided herein, and a T cell signaling domain including, e.g., hinge-CH 2-CH3, a transmembrane domain, and one or more cytoplasmic signaling domains. In certain embodiments, transmembrane domains include, but are not limited to, transmembrane domains from CD8 α, CD28, CD3 ∈, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, and CD 154. In certain embodiments, cytoplasmic signaling domains include, but are not limited to, intracellular costimulatory signaling domains from CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, CD54(ICAM), CD152(CTLA4), CD273(PD-L2), CD274(PD-L1), and CD278(ICOS), as well as major signaling domains from zeta TCR, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 e, CD5, CD22, CD79a, CD79B, and CD66 d.

The disclosure further provides for the use of the CAR so produced in immunotherapy, for example in chimeric antigen receptor T cell therapy (CAR-T).

Also provided herein is a method of generating a Chimeric Antigen Receptor (CAR), the method comprising the step of expressing a first nucleic acid operably linked to a second nucleic acid, wherein the first nucleic acid encodes an antigen binding domain derived from an antibody or antigen binding fragment thereof produced according to the methods described herein, and wherein the second nucleic acid encodes one or more T cell signaling domains.

Immune cells such as T cells and Natural Killer (NK) cells can be genetically engineered to express CARs. T cells expressing CARs are referred to as CAR-T cells. The CAR can mediate antigen-specific cellular immune activity in the T cell, thereby enabling the CAR-T cell to eliminate cells (e.g., tumor cells) that express the target antigen. In one embodiment, binding of a CAR-T cell provided herein to a cancer cell results in proliferation and/or activation of the CAR-T cell, wherein the activated CAT-T cell can release a cytotoxic factor, e.g., cytotoxicity. Perforin, granzyme and granulysin, and initiates cytolysis and/or apoptosis of cancer cells.

In some embodiments, the T cell activation domain of the CAR comprises a costimulatory signaling domain and a TCR signaling domain, which can be linked to each other randomly or in a specified order, optionally with a short peptide, a linker of between, for example, 2 to 10 amino acids in length (e.g., a glycine-serine bimodal linker).

In some embodiments, the CAR further comprises a transmembrane domain. When expressed in a cell, the antigen binding domain is extracellular and the T cell activation domain is intracellular.

In certain embodiments, the CAR comprises an antigen binding domain, a transmembrane domain, a costimulatory signaling region, and a TCR signaling domain, wherein the antigen binding domain specifically binds an antigen and comprises an antigen-binding fragment of an antibody.

The following examples are provided to better illustrate the claimed invention and should not be construed as limiting the scope of the invention. All of the specific compositions, materials and methods described in whole or in part below are within the scope of the invention. These specific compositions, materials and methods are not intended to limit the present invention, but are merely illustrative of specific embodiments that fall within the scope of the invention. Those skilled in the art may develop equivalent compositions, materials, and methods without the exercise of the capabilities of the present invention and without departing from the scope of the present invention. It will be appreciated that many variations in the processes described herein may be made while still remaining within the scope of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.

Examples

Example 1: materials and methods

Materials:

lymphocyte separating medium (MP, cat. V0111A)

LLME: L-leucyl-L-leucine methyl ester (Bachem, cat. G-2550.0001)

Ham's F-12 medium (Gibco, cat.11765047)

Heparin anticoagulant tube (BD, cat.367878)

Disposable blood taking needle (BD, cat.367237)

IL2, interleukin-2, lymphokine, TCGF (biochemical, cat.11848-HNAY1-50)

BCGF-1, BCGF1, BSF-1, BSF1, IL-4, interleukin-4 (sinobiological, cat, GMP-11846-HNAE-100)

CD154, CD40 ligand (sinobiological, cat.10239-H01H-50)

OX40L(sinobiological,cat.13127-H04H-100)

Human ICOS ligand/B7-H2/ICOSLG (Histagg) (biochemical, cat.11559-H08H-100)

Human ICOS/AILIM/CD278 protein (His & Fc Tag) (sinobiological, cat.10344-H03H-100)

Human interleukin-21/IL 21(sinobiological, cat. GMP-10584-HNAE-20)

Human BLyS/TNFSF13B/BAFF (sinobiological, cat.10056-HNCH-5)

epinephrine-B1 (sinobiological, cat.10894-H08H)

Goat anti-human IgG-Fc (HRP) (sinobiological, cat. SSA001-1)

Sheep anti-human IGM μ chain (Abcam, cat. ab97205)

GlutaMAX^TM Supplement(Gibco,cat.35050-061)

MEM NEAA(Gibco,cat.11140-050)

Sodium pyruvate (Gibco, cat.11360-070)

DMEM (glutamine free, sodium pyruvate free, HEPES free) (Gibco, cat.11960-051)

Penicillin-streptomycin, liquid (Gibco, cat.15140122)

Recombinant human insulin (yuanyye, cat. S31559-100mg)

RPMI1640 medium (Gibco, cat.21875091)

DAPI (4', 6-diamino-2-phenylindole; stock solution: 5mg/ml in dH 2O; Thermo Fisher, cat. No. D1306)

TMB chromogenic substrate (TIANGEN, cat. PA107-01)

FBS(GIBCO,cat.10099141)

PBS(8117158)

E6446 dihydrochloride (MCE, cat. HY-12756A)

Anti-human CD3 PE-Cy7(eBioscience, cat. BG-05121-77-100)

Anti-human CD21 PE (eBioscience, cat.85-12-0219-42)

Mouse anti-human CD35-FITC (eBioscience, cat.05-9600-02)

Mouse anti-human CD19 PerCP-Cy5.5(eBioscience, cat. BG-11211-70-100)

Image reader (Biotek, cat. rotation 5)

96-well enzyme label plate (Corning, cat 9018)

Human IL1 beta (Novoprotein, cat. CG93)

Human CCL8(Novoprotein, cat. CC95)

Human CXCL5(Novoprotein, cat. cf14)

Murine CXCL16(Novoprotein, cat. CC44)

Recombinant human CXCL13(SB, cat.10621-HNAE)

Recombinant human CCL4(SB, cat.10899-H08Y)

Recombinant human IL27(SB, cat.10076-H085)

Human CXCL2(Novoprotein, cat. c096)

Chicken OVA (sigma, cat. A5378-1G)

Total RNA extraction kit (Takara, cat.9767)

The method comprises the following steps:

immunization of mice

Ovalbumin (OVA) was used as antigen. Monoclonal antibodies against OVA were developed by sequential immunization of BLAB/C mice. BALB/C mice were immunized by all injections via the footpad injection route. The first and several subsequent injections of OVA per mouse were mixed with Adju-Phos and CpG, and the last injection of DPBS containing 10ug OVA in the absence of adjuvant. For this protocol, BLAB/C mice were immunized on days 0,3, 6, 10, 13, 17, 20 and 24.

Tissue isolation

Mice were anesthetized with isoflurane and the abdomen of those mice was opened under sterile conditions. The abdominal subcutaneous fat layer was collected, the medial lymph nodes were discarded, and the remaining tissues were stored at-20 ℃.

Total RNA extraction from perilymphatic adipose tissue

Mice were immunized with chicken Ovalbumin (OVA) and lymph nodes were harvested 30 days later. Adipose tissue associated with lymph nodes was dissected out. Total RNA was extracted by total RNA isolation kit (Takara, catalog 9767).

RNA-seq

We prepared the sample and sent it to anoroadigomer for RNA sequencing and analyzed the data by R language.

Preparation of human Peripheral Blood Mononuclear Cells (PBMC)

Media for PBMC culture were prepared: (RPMI 1640: DMEM: Ham's F12 ═ 1: 1: 2) (eRDF) was supplemented with 10% FBS. Fresh PBMCs were harvested from several healthy donors (approximately 40 ml/time/person). PBMCs were separated by density gradient centrifugation as previously described in the human monoclonal antibody book. The number of cells was counted using a hemocytometer.

In vitro immunization

PBMCs were washed and diluted with 10% FBS + eRDF. Adjusting cell density to 1X 10⁷Cells/ml, treated with 0.25mM LLME for about 20 minutes. The supernatant was discarded and the cells were resuspended in 10% FBS + eRDF. Adjusting the cell density to 9X 10⁵cells/ml. The cell suspension was transferred to a 96-well plate and 2ug/ml antigen, 10ng/ml IL2, 50ng/ml IL21, CD40L, ICOS, synthetic TLR7/8 agonist were added, respectively. The tissue was cultured at 37 ℃ under 5% CO2 for 7 days. Half of the medium was replaced and the cytokine/activator mixture was added on day 7. Cells were cultured at 37 ℃ under 5% CO2 for 7-21 days. Supernatants were collected at day 7, day 14 or day 21 to analyze antibody production by ELISA, while the pellets were used to test gene expression by PCR or RT-PCR. The collected cells were also used for ELISpot detection and FACS analysis.

Measurement of antibody levels after incubation with antibody enhancing compositions

After day 7 or 14 of addition of cytokine or antibody enhancing composition and antigen, supernatants were harvested and added to antigen OVA coated plates. After 2 hours of incubation, HRP-labeled anti-human IgG or anti-human IgM was added and the amount of antigen-specific antibody was measured using TMB as a substrate. Data represent the average of 2 replicates. Error bars represent SD. One representative data of 3 independent experiments is shown.

Flow analysis

We analyzed stained cells on aireii (bd) and processed the flow cytometer data using FlowJo software (Tree Star). PBMCs were collected into Snap-lock microtubes. For analysis of T cells or B cells, tubes were maintained at 4 ℃ unless otherwise stated. After centrifugation, the cells were washed and resuspended in PBS. For analysis of T follicular helper cells, CD3-FITC (BD), CD4-PerCP-Cy, respectively, were used^TMPBMCs were stained with antibodies CXCR5-PE/Cy7 (Biolegened) and CD45RA-PE (eBioscience) 5.5 (BD). T follicle helper cells were identified using CD3+ CD4+ CXCR5+ CD45 RA-. To analyze GC-like B cells, PBMCs were stained with CD19-PE (eBioscience), GL7-Alexa Fluor 488(eBioscience), Fas-APC (eBioscience) antibodies. GC-like B cells were defined as CD19+ GL7+ Fas +.

Reverse transcription PCR

Quantitative RT-PCR was performed with a BioRad iccycler and relative mRNA expression levels normalized to GAPDH were calculated using the 2- (Δ Δ CT) method.

Enzyme linked immunosorbent assay

ELISA plates were coated with 5. mu.g/ml antigen overnight at 4 ℃ and washed in PBST (containing 0.5% Tween-20). Before adding cell culture supernatant and horseradish peroxidase (HRP) -labeled detection antibody, the plates were blocked with 5% BSA (dilution: dilution ratio of HRP-bound IgG-specific antibody (Jackson) and HRP-labeled IgM-specific antibody was one-half of 2,500.

Enzyme-linked immunospot assay (ELISpot)

Nitrocellulose 96-well MAHAS4510 plates (Millipore) were coated overnight at 4 ℃ with 50mM sodium bicarbonate buffer (pH 9.6) (5 μ g/mL). Plates were washed and blocked with 10% fetal bovine serum in RPMI1640 for 2h at 37 ℃. PBMCs were seeded at 3X 10^5 cells/well and incubated at 37 ℃ for 24 hours. Spot-forming cells (SFC) were then detected using goat anti-human IgG antibody at 2,000 fold dilution coupled with horseradish peroxidase and incubated at 37 ℃ for 2 hours. Antibody binding was assessed by addition of TrueBlue substrate solution substrate (KPL, gaithersburg, maryland).

Statistical tests with appropriate underlying assumptions about the data distribution and variance characteristics are used. Two-way ANOVA was used to compare endpoint means for different groups unless otherwise stated. Regression and mapping were performed using Prism6 (GraphPad).

Example 2: efficiency of antibody production of stimulating factor

In vitro expansion of PBMCs includes antibody-producing B cells, T cells, dendritic cells and adipocytes that can form germinal center-like structures in an in vitro culture system, as described in WO 2018/205917. IL2, IL21, ICOSL, ICOS, CD40L and Toll-like receptor (TLR) agonists have been shown to have stimulatory effects on antibody production in B cells.

Specifically, in an in vitro antibody production system, IL2 and/or IL21 promote the proliferation of PBMCs, including the promotion of B cell, T cell and dendritic cell populations (see fig. 1 and 3 of WO 2018/205917); IL21 also promotes class switching from IgM to IgG (see figures 3A-3B of WO 2018/205917); both ICOSL and ICOS induced antibody production by B cells, while ICOSL and CD40L synergistically enhanced IgG production, rather than ICOSL or CD40L alone (see fig. 2A-2B and 4A-4B of WO 2018/205917).

In addition, TLR agonists (e.g., synthetic TLR7/8 agonists and TLR9 agonists) also serve as key factors in enhanced antibody production, suggesting that TLR7/8 agonists are more potent than CD 40L. Stimulation of anti-OVA antibody production (see FIG. 6A of WO 2018/205917). For IgG antibodies, the TLR7/8 agonist was more effective at 14 days in vitro than at 7 and 21 days in vitro (50nM and 500nM TLR agonist effect 3.5 and 10.0 times higher than CD40L, respectively) (FIG. 6A)

Figure 10 of WO2018/205917 shows that the TLR9 agonist CpG ODN (2 μ g/ml) stimulates production of anti-OVA antibodies similar to the CD40L effect at day 14 after addition of factors (for IgG and IgM).

The expression of AID (involved in B-cell affinity maturation by inducing hypermutations in the antibody gene) and BLIMP-1 (indicating proliferation and differentiation of active B-cells) was increased by TLR7 (imiquimod, 500nM) agonists or TLR7/8 agonists. Both figures 8 and 9 of WO2018/205917 show that TLR7 agonists and synthetic TLR7/8 agonists are far superior to CD40L in stimulating expression of AICDA and BLIMP-1. It is also shown in fig. 8 and 9 of WO2018/205917 that TLR7 has superior ability to enrich for antibody variants by inducing hypermutations and higher than CD 40L.

Figures 11A-11C of WO2018/205917 show that low concentrations of TLR9 antagonist E6446 (e.g., 0.02 μ M and 0.2 μ M) promote the effect of TLR7/8 agonists on antibody IgG, indicating a synergistic effect between TLR7/8 agonists and low concentrations of TLR9 antagonist E6446. However, for IgG and IgM production, high concentrations of E6446(10 μ M) reversed the effect of TLR7/8 agonists in 7-day and 14-day cultures (see fig. 11D-11F of WO 2018/205917).

Figure 11G of WO2018/205917 shows that stimulation of PBMCs with TLR7/8 agonists results in a significant increase in IgG response and cellular activity following in vitro blockade of TLR9 (0.02 μ M and 0.2 μ ME6446 dihydrochloride, respectively), indicating that TLR7/8 agonists promote dendritic cell activation, which is partially reversed by high concentrations of TLR9 antagonist E6446.

Figures 12A-12I of WO2018/205917 show that IL2 and IL21, used in combination with ICOS, CD40L or synthetic TLR7/8 agonists, respectively, produce a synergistic effect, complementarily enhancing IgG production.

FIGS. 13A-13F of WO2018/205917 show that ICOS (24nM, 55nM, 100nM), CD40L (10nM, 24nM, 55nM), TLR7/8 agonist (0.1nM, 50nM, 500nM) modulate IgG and IgM responses. Dose-dependence was present in the presence of 4. mu.g/ml OVA, 10ng/ml IL2 and 50ng/ml IL 21.

Example 3: identification of adipose tissue-derived proteins that enhance antibody production

In searching for adipose tissue-derived proteins that stimulate antibody production, RNA seq was performed and analyzed to identify genes that are up-regulated in adipose tissue associated with lymph nodes following antigen immunization. Total RNA from immunized or non-immunized adipose tissues was processed by RNA-seq technique and then analyzed for differential gene expression.

Using "2-fold increase" as a cutoff criterion, a total of 273 genes were upregulated from adipose tissue after immunization (see fig. 1). Among these genes, 69 genes encode secreted proteins. Detailed analysis shows that these secreted proteins can be divided into three classes: cytokines, chemokines and cell adhesion molecules are listed in table 1.

Table 1 complete list of adipose tissue-derived secreted proteins upregulated after immunization.

Example 4: role of adipose tissue-derived secretory protein (ADSP) in antibody production

Previous work has determined that specific antibodies can be produced in vitro by applying an antigen (e.g., OVA) to human Peripheral Blood Mononuclear Cells (PBMCs) and incubating for one or two weeks in combination with a regulatory factor. Two essential factors are IL2 and IL21 (collectively referred to herein as "basal" or "minimal medium"). ICOS, CD40L, TLR7/8 agonists may further stimulate antibody production. To test the up-regulated ADSPs in table 1, further experiments were performed.

In all experiments performed below, statistical analysis was performed using ANOVA followed by special tests. Data represent the mean of 3 replicates and error bars represent SD. P <0.001, p < 0.0001.

1.1. Efficiency of interleukin

An exemplary cytokine, IL1 β, was tested. PBMCs were incubated in vitro with OVA, basal medium and some known stimulatory factors ICOS, CD40L, TLR7/8 agonist or IL1 β (different doses) (1.5X 10^5 cells/well, 96 well plates). After 7 or 14 days, the supernatants were collected and the production of IgG/IgM antibodies was measured by ELISA assay. Figure 2 shows representative data from 3 independent experiments. In general, the effect of IL1 β in stimulating antibody production was similar to that of TLR7/8 agonists, but better than ICOS and CD 40L.

1.2. Efficiency of chemokines

1.2.1CCL8

An experiment was performed exactly as described in section 1.1 of example 3, except CCL8 was used instead of IL1 β (see fig. 3). For IgG production, CCL8 did not function as well as the TLR7/8 agonist, but better than ICOS and CD 40L.

1.2.2CXCL5

An experiment was performed exactly as described in section 1.1 of example 3, except that CXCL5 was used instead of IL1 β (see fig. 4). For IgG cultured for 14 days in vitro, the effect of CXCL5 was dose-dependent, with CXCL5 being less effective than the TLR7/8 agonist but better than ICOS and CD40L at 20 ng/ml.

1.3 efficiency of other ADSPs

The experiment described in section 1.1 of example 3 was exactly the same except that CXCL13, CCL4, IL27, CXCL16, CXCL2 were used instead of IL1 β, respectively, see fig. 5. CCL4, IL27, CXCL16, CXCL2 were more effective than Basic alone for 14 days of in vitro IgG production. For IgM, CXCL13, CCL4, IL27, CXCL16 produced in vitro for 14 days, the effect was superior to Basic alone.

Example 5: in vitro immunoassay, antigen-specific antibodies produced in the presence of ADSP

To investigate the independent function of components in the immune system in vitro, exactly the same experiment was performed as in section 1.1 of example 3. Cultured PBMCs were treated with IL-1 β, CCL8, CXCL5 or the TLR7/8 agonist R848 (1.5X 10^5 cells/well, 96-well plate) for 14 days. The media were harvested and the production of OVA-specific IgG (fig. 6A) or IgM (fig. 6B) antibodies was measured by ELISA assay. Nonspecific antibodies were measured by BSA-coated ELISA plates to determine if and how much they bound to BSA (fig. 6C). Representative data for 3 independent experiments are shown. Statistics data: analysis of variance (ANOVA), followed by post hoc analysis. Data represent the mean of 3 replicates and error bars represent SEM. P <0.01, p < 0.0001.

Based on the antibodies produced in the presence or absence of antigen OVA (anti-OVA IgG, anti-OVA IgM, and anti-BSA IgG), the antibodies produced under "minimal medium" conditions are those produced in the absence of antigen stimulation, indicating that they are non-specific, and that "minimal medium" may non-specifically activate antibody-producing B cells (see "minimal medium alone" in fig. 6A-6C), the high level of IL-1 β may promote the production of anti-OVA IgG, compared to anti-OVA IgM and anti-BSA IgG in the presence of antigen OVA, indicating that IL-1 β specifically stimulates the activation of OVA-specific B cells (i.e., antigen-specific B cells) (see fourth panels "minimal + OVA". CCL8 and CXCL5 in fig. 6A-6C, in the production of anti-OVA IgG and anti-OVA IgM antibodies, activation of OVA-specific B cells can be similarly stimulated.

Example 6: expression of ADSPS in mice

This experiment was performed to investigate the expression levels of IL-1 β, CCL8, CXCL5, IL36 in lymph node derived adipose tissue with or without OVA immunization (10 μ g/time, 8 times, see fig. 7). Quantitative analysis was performed by quantitative RT-PCR and 2- (Δ Δ OT) methods to determine mRNA levels for different factors. Adipose tissue was collected from healthy immunized (sample) or non-immunized (control) OVA BALB/C mice. ACTB expression was used as an internal control. Representative data for 3 independent experiments are shown. In fig. 7, statistical analysis was performed using the ANOVA test. P < 0.0001.

While the present disclosure has been particularly shown and described with reference to particular embodiments, some of which are preferred, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the disclosure.

Claims

1. A method of producing an antibody or antigen-binding fragment thereof that specifically binds to an antigen of interest, the method comprising

(ii) incubating the mixture in the presence of a nutrient,

2. The method of claim 1, wherein the antibody enhancing composition further comprises IL2 and/or IL 21.

3. The method of claim 1, wherein the adipose tissue-derived secreted protein comprises one or more cytokines and/or one or more cell adhesion molecules.

4. The method of claim 3, wherein the cytokines comprise one or more interleukins and/or one or more chemokines.

5. The method of claim 4, wherein the interleukin is selected from the group consisting of IL-1 β, IL1f9, IL10, IL27, IL33, and IL18 BP.

6. The method of claim 4, wherein said chemokine comprises one or more CC chemokines selected from CCL4, CCL8, CCL6, CCL9, and CCL11, or one or more CXC chemokines selected from CXCL2, CXCL5, CXCL16, CXCL9, and CXCL 13.

7. The method of claim 3 or 4, wherein the cytokine is selected from the group consisting of IL-1 β, CCL8, and CXCL 5.

8. The method of claim 3, wherein the cell adhesion molecule is selected from ICAM1, CSF3r, Itgam, Siglecf, Adam8, Chl, Sirpa, Nrcam, Emilin2, Emilin1, Tubb6, and/or Parvb.

9. The method of any one of claims 1 to 8, wherein the ADSPs are derived from adipose tissue.

10. The method of any one of claims 1-9, wherein the AGC comprises at least one B cell and at least one T follicular helper cell.

11. The method of any one of claims 1-9, wherein the AGC comprises at least one B cell and at least one dendritic cell.

12. The method of any one of claims 1 to 9, wherein the AGC comprises at least one B cell, at least one T follicular helper cell, and at least one dendritic cell.

13. The method of any one of claims 1 to 12, wherein the AGC further comprises at least one adipocyte.

14. The method of any one of claims 1-13, wherein the AGC comprises PBMCs.

15. The method of claim 14, wherein the PBMCs are isolated from a blood sample derived from human Hematopoietic Stem Cells (HSCs), from induced pluripotent stem cells (ipscs), or from umbilical cord blood.

16. The method of any one of the preceding claims, wherein the antibody-enhancing composition further comprises a co-stimulatory agent, a toll-like receptor (TLR) agonist, a CpG oligodeoxynucleotide (CpG ODN), an anti-apoptotic protein, TNF, Interferon (INF), a lipid, avasimid, EFNB1, EPHB4, protein B2, axon-targeting protein 4C, BLIMP-1, IRF4, or any combination thereof.

17. The method of claim 16, wherein the co-stimulus comprises CD40, CD40L, ICOSL, ICOS, APRIL, B cell activating factor of the TNF family (BAFF), OX40, and/or OX 40L.

18. The method of claim 16, wherein the CpG ODN comprises CpG2006 and/or D/KCpG.

19. The method of claim 16, wherein the anti-apoptotic protein comprises Bcl-2, Bcl-6, Bcl-XL, Bcl-w, Mcl-1, and/or an analog thereof.

20. The method of claim 16, wherein the TLR agonist comprises a TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a TLR8 agonist, a TLR7/8 agonist, and/or a TLR9 agonist.

21. The method of any one of the preceding claims, wherein the adipose tissue-derived secreted protein enhances antibody production via AGC, enhances activation and differentiation of B cells in AGC, and/or enhances maturation of B cells in AGC.

22. The method of any one of the preceding claims, further comprising isolating antibodies produced in the mixture.

23. The method of claim 22, further comprising obtaining a nucleic acid sequence encoding a variable region of the antibody.

24. The method of claim 23, further comprising introducing the nucleic acid sequence into a host cell under conditions suitable for expression of the antibody or antigen-binding fragment thereof.

25. The method of any one of the preceding claims, further comprising assessing whether the antibody specifically binds to the antigen of interest.

26. The method of any one of the preceding claims, wherein the ADSP is present at a concentration of at least 1ng/ml, 10ng/ml or 50 ng/ml.

27. The method of claim 2, wherein IL2 is present at a concentration of at least 10 ng/ml.

28. The method of claim 2, wherein IL21 is present at a concentration of at least 50 ng/ml.

29. The method of claim 26, wherein the ADSPs are present for at least 1 day.

30. The method of claim 27, wherein the IL2 is present for at least 1 day.

31. The method of claim 28, wherein the IL21 is present for at least 1 day.

32. A method of inducing proliferation of an antibody-producing cell composition (AGC), B cell activation and differentiation, B cell maturation, and/or promoting class switching in AGC to produce IgG, wherein the method comprises culturing the AGC in a medium comprising IL2, adipose tissue-derived secreted protein (ADSP), and/or IL 21.

33. The method of any one of the preceding claims, wherein the antibody is a fully human monoclonal antibody.

34. A method of producing an antibody or antigen-binding fragment thereof that specifically binds to an antigen of interest, the method comprising:

(ii) incubating the mixture in the presence of a nutrient,

35. The method of claim 34, further comprising

Obtaining nucleic acid molecules encoding the variable regions of the antibodies from the mixture; and optionally

Introducing the nucleic acid molecule into a host cell under conditions suitable for expression of the antibody or antigen-binding fragment thereof.

36. The method of claim 35, further comprising isolating antibodies or antigen-binding fragments thereof secreted by the host cell.

37. A composition comprising an isolated antibody producing cell composition (AGC) comprising at least one B cell and at least one other type of Peripheral Blood Mononuclear Cell (PBMC) -derived cell, an antibody enhancing composition and a culture medium.

38. The composition of claim 37, further comprising an antigen of interest.

39. The composition of claim 37, wherein the antibody enhancing composition further comprises IL2 and/or IL 21.

40. The composition of claim 37, wherein the AGC comprises at least one B cell and at least one T follicle helper cell.

41. The composition of claim 37, wherein the AGC comprises at least one B cell and at least one dendritic cell.

42. The composition of claim 37, wherein the AGC comprises at least one B cell, at least one T follicle helper cell, and at least one dendritic cell.

43. The composition of claim 37, wherein the AGC comprises PBMCs.

44. The composition of any one of claims 40-43, wherein the AGC further comprises at least one adipocyte.

45. The composition of claim 37, wherein the antibody enhancing composition comprises one or more antibody enhancing factors selected from ADSP, CD40L, ICOSL, ICOS, TLR agonists, and any combination thereof.

46. A method of identifying an antibody enhancing factor for in vitro immunization comprising:

c) culturing PBMCs in a culture medium comprising the antigen of interest, IL2, IL21 and the protein;

47. The method of claim 46, wherein the cell is an adipocyte, T follicular helper cell, B cell, or dendritic cell.

48. The method of claim 46, wherein the protein is expressed by adipocytes, T follicular helper cells, B cells, or dendritic cells.

49. A method of generating a Chimeric Antigen Receptor (CAR), the method comprising the step of expressing a first nucleic acid operably linked to a second nucleic acid, wherein the first nucleic acid encodes an antigen binding domain derived from an antibody or antigen binding fragment thereof generated according to the method of any one of claims 1 to 36, and wherein the second nucleic acid encodes a T cell signaling domain.

50. A method of treating cancer in a subject, comprising:

expressing in a T cell a first nucleic acid operably linked to a second nucleic acid, wherein the first nucleic acid encodes an antigen binding domain derived from an antibody or antigen binding fragment thereof produced according to the method of any one of claims 1 to 36, and wherein the second nucleic acid encodes a T cell signaling domain; and

administering the T cell to the subject.

51. The method of claim 50, wherein the T cell is obtained from the subject.